Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

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Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

Acquisition of a Novel Sulfur-Oxidizing Symbiont in the Gutless Marine Worm Inanidrilus exumae

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2018-01-01

ABSTRACT Gutless phallodrilines are marine annelid worms without a mouth or gut, which live in an obligate association with multiple bacterial endosymbionts that supply them with nutrition. In this study, we discovered an unusual symbiont community in the gutless phallodriline Inanidrilus exumae that differs markedly from the microbiomes of all 22 of the other host species examined. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization revealed that I. exumae harbors cooccurring gamma-, alpha-, and deltaproteobacterial symbionts, while all other known host species harbor gamma- and either alpha- or deltaproteobacterial symbionts. Surprisingly, the primary chemoautotrophic sulfur oxidizer “Candidatus Thiosymbion” that occurs in all other gutless phallodriline hosts does not appear to be present in I. exumae. Instead, I. exumae harbors a bacterial endosymbiont that resembles “Ca. Thiosymbion” morphologically and metabolically but originates from a novel lineage within the class Gammaproteobacteria. This endosymbiont, named Gamma 4 symbiont here, had a 16S rRNA gene sequence that differed by at least 7% from those of other free-living and symbiotic bacteria and by 10% from that of “Ca. Thiosymbion.” Sulfur globules in the Gamma 4 symbiont cells, as well as the presence of genes characteristic for autotrophy (cbbL) and sulfur oxidation (aprA), indicate that this symbiont is a chemoautotrophic sulfur oxidizer. Our results suggest that a novel lineage of free-living bacteria was able to establish a stable and specific association with I. exumae and appears to have displaced the “Ca. Thiosymbion” symbionts originally associated with these hosts. IMPORTANCE All 22 gutless marine phallodriline species examined to date live in a highly specific association with endosymbiotic, chemoautotrophic sulfur oxidizers called “Ca. Thiosymbion.” These symbionts evolved from a single common ancestor and represent the ancestral trait for

SV40 Assembly In Vivo and In Vitro

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Ariella Oppenheim

2008-01-01

Full Text Available The Simian virus 40 (SV40 capsid is a T = 7d icosahedral lattice ∼45 nm in diameter surrounding the ∼5 kb circular minichromosome. The outer shell is composed of 360 monomers of the major capsid protein VP1, tightly bound in 72 pentamers. VP1 is a jellyroll β-barrel, with extending N- and C-terminal arms. The N-terminal arms bind DNA and face the interior of the capsid. The flexible C-arms tie together the 72 pentamers in three distinct kinds of interactions, thus facilitating the formation of a T = 7 icosahedron from identical pentameric building blocks. Assembly in vivo was shown to occur by addition of capsomers around the DNA. We apply a combination of biochemical and genetic approaches to study SV40 assembly. Our in vivo and in vitro studies suggest the following model: one or two capsomers bind at a high affinity to ses, the viral DNA encapsidation signal, forming the nucleation centre for assembly. Next, multiple capsomers attach concomitantly, at lower affinity, around the minichromosome. This increases their local concentration facilitating rapid, cooperative assembly reaction. Formation of the icosahedron proceeds either by gradual addition of single pentamers to the growing shell or by concerted assembly of pentamer clusters.

A monoclonal antibody against SV40 large T antigen (PAb416) does not label Merkel cell carcinoma.

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Pelletier, Daniel J; Czeczok, Thomas W; Bellizzi, Andrew M

2018-07-01

Merkel cell carcinoma represents poorly differentiated neuroendocrine carcinoma of cutaneous origin. In most studies, the vast majority of Merkel cell carcinomas are Merkel cell polyomavirus (MCPyV)-associated. SV40 polyomavirus immunohistochemistry is typically used in the diagnosis of other polyomavirus-associated diseases, including tubulointerstitial nephritis and progressive multifocal leukoencephalopathy, given cross-reactivity with BK and JC polyomaviruses. MCPyV-specific immunohistochemistry is commercially available, but, if antibodies against SV40 also cross-reacted with MCPyV, that would be advantageous from a resource-utilisation perspective. Tissue microarrays were constructed from 39 Merkel cell carcinomas, 24 small-cell lung carcinomas, and 18 extrapulmonary visceral small-cell carcinomas. SV40 large T antigen immunohistochemistry (clone PAb416) was performed; MCPyV large T antigen immunohistochemistry (clone CM2B4) had been previously performed. UniProt was used to compare the amino acid sequences of the SV40, BK, JC and MCPyV large T antigens, focusing on areas recognised by the PAb416 and CM2B4 clones. SV40 immunohistochemistry was negative in all tumours; MCPyV immunohistochemistry was positive in 38% of Merkel cell carcinomas and in 0% of non-cutaneous poorly differentiated neuroendocrine carcinomas. UniProt analysis revealed a high degree of similarity between SV40, BK, and JC viruses in the region recognised by PAb416. There was less homology between SV40 and MCPyV in this region, which was also interrupted by two long stretches of amino acids unique to MCPyV. The CM2B4 clone recognises a unique epitope in one of these stretches. The PAb416 antibody against the SV40 large T antigen does not cross-react with MCPyV large T antigen, and thus does not label Merkel cell carcinoma. © 2018 John Wiley & Sons Ltd.

Simian virus 40 inhibits differentiation and maturation of rhesus macaque DC-SIGN+-dendritic cells

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Changyong G

2010-09-01

Full Text Available Abstract Dendritic cells (DC are the initiators and modulators of the immune responses. Some species of pathogenic microorganisms have developed immune evasion strategies by controlling antigen presentation function of DC. Simian virus 40 (SV40 is a DNA tumor virus of rhesus monkey origin. It can induce cell transformation and tumorigenesis in many vertebrate species, but often causes no visible effects and persists as a latent infection in rhesus monkeys under natural conditions. To investigate the interaction between SV40 and rhesus monkey DC, rhesus monkey peripheral blood monocyte-derived DC were induced using recombinant human Interleukin-4 (rhIL-4 and infective SV40, the phenotype and function of DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN+ DC were analyzed by flow cytometry (FCM and mixed lymphocyte reaction (MLR. Results showed that SV40 can down-regulate the expression of CD83 and CD86 on DC and impair DC-induced activation of T cell proliferation. These findings suggest that SV40 might also cause immune suppression by influencing differentiation and maturation of DC.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

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Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Host-cell reactivation of ultraviolet-irradiated SV 40 DNA in five complementation groups of xeroderma pigmentosum

International Nuclear Information System (INIS)

Abrahams, P.J.; Eb, A.J. van der

1976-01-01

Host-cell reactivation of UV-irradiated double-stranded SV40 DNA was studied in BSC-1 monkey cells, normal human cells, heterozygous Xeroderma pigmentosum xp cells, representative cell strains of the five complemention groups of XP and in XP 'variant' cells. The following percentages of survival of the plaque-forming ability of double-stranded SV40 DNA were found in XP cells compared with the value found in normal monkey and human cells: groupA, 13%; group B, 30%; group C, 18%; group D, 14%; group E, 59%; and in the heterozygous XP cells almost 100%. The survival in XP 'variant' cells was 66%. The survival of single-stranded SV40 DNA in BSC-1 cells was much lower than that of double-stranded SV40 DNA in XP cells of complementation group A, which possibly indicates that some repair of UV damage occurs even in XP cells of group A

Early superoxide dismutase alterations during SV40-transformation of human fibroblasts.

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Bravard, A; Hoffschir, F; Sabatier, L; Ricoul, M; Pinton, A; Cassingena, R; Estrade, S; Luccioni, C; Dutrillaux, B

1992-11-11

The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts

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Easton Marilyn J

2010-02-01

Full Text Available Abstract Background Simian Virus 40 (SV40 immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC or SV40-immortalized and then 3-MC-transformed (HUC-TC. Results To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-γ and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-γ metabolic responses were functional in both cell lines, but IFN-γ anti-proliferative responses functioned only in tumor cells. Conclusions Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

Impaired CK1 delta activity attenuates SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

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Heidrun Hirner

Full Text Available Simian virus 40 (SV40 is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA

The mechanism of thioacetamide-induced apoptosis in the L37 albumin-SV40 T-antigen transgenic rat hepatocyte-derived cell line occurs without DNA fragmentation.

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Bulera, S J; Sattler, C A; Gast, W L; Heath, S; Festerling, T A; Pitot, H C

1998-10-01

The hepatotoxicant thioacetamide (TH) has classically been used as a model to study hepatic necrosis; however, recent studies have shown that TH can also induce apoptosis. In this report we demonstrate that 2.68+/-0.54% of the albumin-SV40 T-antigen transgenic rat hepatocytes undergo TH-induced apoptosis, a level comparable to other in vivo models of liver apoptosis. In addition, TH could induce apoptosis and necrosis in the L37 albumin-SV40 T-antigen transgenic rat liver-derived cell line. Examination of dying L37 cells treated with 100 mM TH by electron microscopy revealed distinct morphological characteristics that could be attributed to apoptosis. Quantitation of apoptosis by FACS analysis 24 h after treatment with 100 mM TH revealed that 81.3+/-1.6% of the cells were undergoing apoptosis. In contrast, when L37 cells were treated with 250 mM TH, cells exhibited characteristics consistent with necrotic cell death. DNA fragmentation ladders were produced by growth factor withdrawal-induced apoptosis; however, in 100 mM TH-induced apoptosis, DNA fragmentation ladders were not observed. Analysis of endonuclease activity in L37 cells revealed that the enzymes were not inactivated in the presence of 100 mM TH. The data presented in this report indicate that the L37 cell line could be used to study the mechanism of TH-induced apoptosis that was not mediated through a mechanism requiring DNA fragmentation.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

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Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

International Nuclear Information System (INIS)

Su, L.-N.; Little, J.B.

1992-01-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author)

SV40 large T-p53 complex: evidence for the presence of two immunologically distinct forms of p53

International Nuclear Information System (INIS)

Milner, J.; Gamble, J.

1985-01-01

The transforming protein of SV40 is the large T antigen. Large T binds a cellular protein, p53, which is potentially oncogenic by virtue of its functional involvement in the control of cell proliferation. This raises the possibility that p53 may mediate, in part, the transforming function of SV40 large T. Two immunologically distinct forms of p53 have been identified in normal cells: the forms are cell-cycle dependent, one being restricted to nondividing cells (p53-Go) and the second to dividing cells (p53-G divided by). The authors have now dissociated and probed the multimeric complex of SV40 large T-p53 for the presence of immunologically distinct forms of p53. Here they present evidence for the presence of p53-Go and p53-G divided by complexed with SV40 large T

Inhibition of in vitro SV40 DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Gough, G.; Wood, R.W.

1989-01-01

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble humancell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m 2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m 2 . The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication (author). 21 refs.; 2 figs

Hyperacetylation and differential deacetylation of histones H4 and H3 define two distinct classes of acetylated SV40 chromosomes early in infection

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Milavetz, Barry

2004-01-01

SV40 chromosomes undergoing encapsidation late in infection and SV40 chromatin in virions are hyperacetylated on histones H4 and H3. However, the fate of the SV40 chromosomes containing hyperacetylated histones in a subsequent round of infection has not been determined. In order to determine if SV40 chromosomes undergo changes in the extent of histone acetylation during early infection, we have analyzed SV40 chromosomes isolated 30 min and 3 h postinfection by quantitative ChIP assays, depletion ChIP assays, competitive ChIP assays, and ChIP assays combined with restriction endonuclease sensitivity using antibodies to hyperacetylated histones H4 and H3. We have shown that at 30 min postinfection, the hyperacetylated histones are associated with two distinct classes of SV40 chromosomes. One form is hyperacetylated specifically on histone H4 while a second form is hyperacetylated on both H4 and H3. Both forms of chromosomes appear to contain a nucleosome-free promoter region. Over the course of the next few hours of infection, the class of SV40 chromosomes hyperacetylated on only H4 is reduced or completely eliminated through deacetylation

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

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Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

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Rinehart, C A; Mayben, J P; Butler, T D; Haskill, J S; Kaufman, D G

1992-01-01

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.

Effect of caffeine on the ultraviolet light induction of SV40 virus from transformed hamster cells

International Nuclear Information System (INIS)

Zamansky, G.B.; Kleinman, L.F.; Little, J.B.; Black, P.H.; Kaplan, J.C.

1976-01-01

The effect of caffeine on the uv light induction of SV40 virus from two transformed hamster cell lines heterogeneous for the induction of infectious virus was studied. The amount of virus induced was significantly increased in both cell lines when exposure to uv light was followed by treatment with caffeine. Caffeine in the absence of uv irradiation did not stimulate virus induction, nor did it stimulate SV40 replication in a lytic infection. There was an apparent difference in the concentrations of caffeine which maximally stimulated SV40 virus induction in the two cell lines. This effect could not be explained by differences in cell survival after exposure to uv light and caffeine. Since caffeine is known to cause the accumulation of gaps formed in DNA during postreplication repair of uv-irradiated rodent cells, our results support the hypothesis that the formation of gaps or breaks in DNA is an important early step in virus induction

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

International Nuclear Information System (INIS)

LingNah Su; Little, J.B.

1992-01-01

A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

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Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

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Gregory A Sowd

2014-12-01

Full Text Available Simian virus 40 (SV40 and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK(cs kinase activity, facilitates some aspects of double strand break (DSB repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR and do not colocalize with non-homologous end joining (NHEJ factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK(cs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5' to 3' end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

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C.B. Sacramento

2010-08-01

Full Text Available The main objective of the present study was to find suitable DNA-targeting sequences (DTS for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS and hypoxia-responsive element (HRE sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF. The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2 and hypoxia (less than 5% O2 were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Is the standard dose rate for de-contamination, 0.23 mc-Sv/hr, truly 1 mSv/year?

International Nuclear Information System (INIS)

Furuta, Sadaaki

2014-01-01

Examined is the validity of the standard dose rates in the title, which have been additionally defined by Ministry of the Environment (ME) to the measures of Fukushima Nuclear Power Plant Accident. The standard 0.23 mc-Sv/hr is the sum of natural ambient dose rate 0.04 mc-Sv/hr in average of Japan as measured with NaI scintillation surveymeter, plus additional accidental exposure dose 1 mSv/y, which is defined equivalent to 0.19 mc-Sv/hr. Here, the equation of addition 0.04+0.19 mc-Sv/hr is not exactly correct because the unit of each dose value has different means as follows. The natural dose is derived from the value 5.8 mc-R/hr (0.038 mc-Sv/hr, effective dose) of the average national natural dose data (2011) of Ministry of Education, Culture, Sports, Science and Technology. However, if the measurement is done with the surveymeter which practically gives 1 cm dose equivalent, the rate is calculated to be 0.06 mc-Sv/hr. When the Fukushima prefectural natural dose rate 6.4 mc-R/hr is employed, the calculation gives 0.07 mc-Sv/hr. ME defines the additional doses to be 0.19 mc-Sv/hr and 1 mSv/y, which are derived from the calculation: (0.19 mc-Sv/hr x 8 hr outdoor + 0.19 mc-Sv/hr x 0.4 indoor, shielded) x 365 d/y= 1 mSv/y. The dose is assumed to be measurable with the surveymeter (1 cm dose equivalent) and thereby calculation, if the source is mainly "1"3"4Cs and "1"3"7Cs, gives the effective dose of 0.32 mc-Sv/hr to be managed by the meter. As this, the effective dose unit and 1 cm equivalent dose unit are used for calculation of the administrative standard dose rate of 1 mSv/y, which should be recognized by both radiological experts and administration for the explanation to general public. (T.T.)

SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.

Science.gov (United States)

Raghava, Smita; Giorda, Kristina M; Romano, Fabian B; Heuck, Alejandro P; Hebert, Daniel N

2013-06-04

Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.

Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions

International Nuclear Information System (INIS)

Brown, T.C.; Cerutti, P.A.

1986-01-01

The most UV sensitive region within the SV40 viral genome contains the transcriptional promotors and enhancers for the early and late viral genes plus part of the origin of DNA replication. Lesions within this regulatory region are 3.2-fold more effective in inactivating viral DNA than is the same amount of damage randomly distributed throughout the viral genome. The region least sensitive to damage lies within the coding portion of the viral coat protein genes, which are expressed only late in infection and would therefore be transcribed from undamaged progeny viral genomes, provided DNA replication occurs. Damage within this region is only 45% as effective in inactivating viral DNA as are randomly distributed lesions. Thus there is a 7-fold difference in the lethal effect of DNA damage within the most and least sensitive regions of the viral genome. Intermediate sensitivities are observed within the transcribed portion of the viral A gene, coding for the T antigen whose expression is required early in infection, and in a region at the terminus of DNA replication. The sum of the individual sensitivities for all regions of the SV40 genome is equal to the total sensitivity of viral DNA subjected to random damage. (author)

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

Science.gov (United States)

Bogacheva, Mariia; Egorova, Anna; Slita, Anna; Maretina, Marianna; Baranov, Vladislav; Kiselev, Anton

2017-11-01

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Oncogenic transformation of rat lung epithelioid cells by SV40 DNA and restriction enzyme fragments

International Nuclear Information System (INIS)

Daya-Grosjean, L.; Lasne, C.; Nardeux, P.; Chouroulinkov, I.; Monier, R.

1979-01-01

Rat epithelioid lung cells were transformed with various preparations of SV40 DNA using the Ca 2+ -precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic newborn animals or in adult nude mice. One particular transformed line, which had received the HpaII/BamHIA (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the HpaII/BamHIB (41 per cent fragment). This line which grew to high saturatio densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the HpaII/BamHIA fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epithelioid lung cells. (author)

Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1

International Nuclear Information System (INIS)

Mizoguchi, Izuru; Ooe, Yoshihiro; Hoshino, Shigeki; Shimura, Mari; Kasahara, Tadashi; Kano, Shigeyuki; Ohta, Toshiko; Takaku, Fumimaro; Nakayama, Yasuhide; Ishizaka, Yukihito

2005-01-01

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application

pSv3neo transfection and radiosensitivity of human cancer cell lines

International Nuclear Information System (INIS)

Parris, C.N.; Masters, J.R.W.; Green, M.H.L.

1990-01-01

Immortalisation of human fibroblasts by transfection with a plasmid, pSV3neo, results in an increase in their radioresistance. The change in radiosensitivity may either be a consequence of transformation or due to expression of the SV40 T-antigen in pSV3neo. To investigate these two possibilities, we transfected pSV3neo into cells already transformed and immortalised. The radiosensitivies of three human bladder cancer cell lines were unaltered in clones expressing T-antigen, indicating that the changes observed in fibroblasts probably are a consequence of transformation, and not the presence of SV40 T-antigen. (author)

Simvastatin (SV) metabolites in mouse tissues

International Nuclear Information System (INIS)

Duncan, C.A.; Vickers, S.

1990-01-01

SV, a semisynthetic analog of lovastatin, is hydrolyzed in vivo to its hydroxy acid (SVA), a potent inhibitor of HMG CoA reductase (HR). Thus SV lowers plasma cholesterol. SV is a substrate for mixed function oxidases whereas SVA undergoes lactonization and β-oxidation. Male CD-1 mice were dosed orally with a combination of ( 14 C)SV and ( 3 H)SVA at 25 mg/kg of each, bled and killed at 0.5, 2 and 4 hours. Labeled SV, SVA, 6'exomethylene SV (I), 6'CH 2 OH-SV (II), 6'COOH-SV (III) and a β-oxidized metabolite (IV) were assayed in liver, bile, kidneys, testes and plasma by RIDA. Levels of potential and active HR inhibitors in liver were 10 to 40 fold higher than in other tissues. II and III, in which the configuration at 6' is inverted, may be 2 metabolites of I. Metabolites I-III are inhibitors of HR in their hydroxy acid forms. Qualitatively ( 14 C)SV and ( 3 H)SVA were metabolized similarly (consistent with their proposed interconversion). However 3 H-SVA, I-III (including hydroxy acid forms) achieved higher concentrations than corresponding 14 C compounds (except in gall bladder bile). Major radioactive metabolites in liver were II-IV (including hydroxy acid forms). These metabolites have also been reported in rat tissues. In bile a large fraction of either label was unidentified polar metabolites. The presence of IV indicated that mice (like rats) are not good models for SV metabolism in man

Pleomorphic adenoma cells vary in their susceptibility to SV40 transformation depending on the initial karyotype.

Science.gov (United States)

Kazmierczak, B; Thode, B; Bartnitzke, S; Bullerdiek, J; Schloot, W

1992-07-01

Chromosomal aberrations involving 8q12 or 12q13-15 characterize two cytogenetic subgroups of salivary gland pleomorphic adenomas. As the tumors of the two groups differ in their clinical and histologic characteristics, we decided to determine their susceptibility to SV40 transformation. We transfected cell cultures from 13 adenomas with aberrations involving 8q12 and from seven adenomas with involvement of 12q13-15 using an SV40 plasmid coding for the early region of the viral genome. Whereas all cultures with aberrations of 12q13-15 showed transformed foci, only 4 of the 13 cultures with 8q12 abnormalities showed foci of transformed cells. We also observed a much higher immortalization rate in the first group (3/7 vs. 1/13). All successfully transformed tumor cell cultures showed a relatively stable karyotype in the pre-crisis stage and a high mitotic index, were T-antigen positive, and had an extended life span in vitro.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

Science.gov (United States)

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

Science.gov (United States)

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

African Journals Online (AJOL)

In the present work recombinant proteins were produced for used in LUMINEX in order to undergo serological study of Tunisian female population. HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids ...

Bacterial endosymbioses of gutless tube-dwelling worms in nonhydrothermal vent habitats.

Science.gov (United States)

Naganuma, Takeshi; Elsaied, Hosam E; Hoshii, Daiki; Kimura, Hiroyuki

2005-01-01

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to gamma-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of alpha-, beta-, gamma-, and epsilon-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to beta-proteobacteria. An unidentified pogonophoran from the world's deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

DEFF Research Database (Denmark)

Götz, C; Koenig, M G; Issinger, O G

1995-01-01

by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

STX140, but not paclitaxel, inhibits mammary tumour initiation and progression in C3(1/SV40 T/t-antigen transgenic mice.

Directory of Open Access Journals (Sweden)

Florence Meyer-Losic

Full Text Available Despite paclitxael's clinical success, treating hormone-refractory breast cancer remains challenging. Paclitaxel has a poor pharmacological profile, characterized by a low therapeutic index (TIX caused by severe dose limiting toxicities, such as neutropenia and peripheral neuropathy. Consequently, new drugs are urgently required. STX140, a compound previously shown to have excellent efficacy against many tumors, is here compared to paclitaxel in three translational in vivo breast cancer models, a rat model of peripheral neuropathy, and through pharmacological testing. Three different in vivo mouse models of breast cancer were used; the metastatic 4T1 orthotopic model, the C3(1/SV40 T-Ag model, and the MDA-MB-231 xenograft model. To determine TIX and pharmacological profile of STX140, a comprehensive dosing regime was performed in mice bearing MDA-MD-231 xenografts. Finally, peripheral neuropathy was examined using a rat plantar thermal hyperalgesia model. In the 4T1 metastatic model, STX140 and paclitaxel significantly inhibited primary tumor growth and lung metastases. All C3(1/SV40 T-Ag mice in the control and paclitaxel treated groups developed palpable mammary cancer. STX140 blocked 47% of tumors developing and significantly inhibited growth of tumors that did develop. STX140 treatment caused a significant (P<0.001 survival advantage for animals in early and late intervention groups. Conversely, in C3(1/SV40 T-Ag mice, paclitaxel failed to inhibit tumor growth and did not increase survival time. Furthermore, paclitaxel, but not STX140, induced significant peripheral neuropathy and neutropenia. These results show that STX140 has a greater anti-cancer efficacy, TIX, and reduced neurotoxicity compared to paclitaxel in C3(1/SV40 T-Ag mice and therefore may be of significant benefit to patients with breast cancer.

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

International Nuclear Information System (INIS)

Rainbow, A.J.

1989-01-01

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

Safety of recombinant human platelet-derived growth factor-BB in Augment® Bone Graft

Directory of Open Access Journals (Sweden)

Luis A Solchaga

2012-12-01

Full Text Available This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment® Bone Graft (Augment. Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125

International Nuclear Information System (INIS)

Ehrfeld, A.; Planas-Bohne, F.; Luecke-Huhle, C.

1986-01-01

Iodine-125, in the form of 5-[ 125 I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125 I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125 I atoms

Rat bone marrow progenitor cells transduced in situ by rSV40 vectors differentiate into multiple central nervous system cell lineages.

Science.gov (United States)

Louboutin, Jean-Pierre; Liu, Bianling; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

2006-12-01

Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG(+) throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG(+) cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells.

SvABA

DEFF Research Database (Denmark)

Wala, Jeremiah A; Bandopadhayay, Pratiti; Greenwald, Noah

2018-01-01

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection...... due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated Sv...... complex somatic rearrangements with chains of short (applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we...

Transcriptional repression is epigenetically marked by H3K9 methylation during SV40 replication

OpenAIRE

Kallestad, Les; Christensen, Kendra; Woods, Emily; Milavetz, Barry

2014-01-01

Background We have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone m...

Improved AMD counters quicky type to achieve a level of 0.2 log derivative mSv output in counts refills; Mejora de la AMD en contadores tipo quicky para conseguir un nivel derivado de registro de 0,2 mSV en contajes de salida en recargas

Energy Technology Data Exchange (ETDEWEB)

Bravp Perez-Tinao, B.; Marchena Gonzalez, P.; Sollet Danudo, E.

2011-07-01

To ensure that no worker receives contract exposed by internal exposure dose computed not exceed 1 mSv / year, the Nuclear Security Council requested the Spanish nuclear power plants and Tecnatom that in 2010 would ensure a level derived from registration in internal dosimetry counts refills output equal to or less than 0.2 mSv, which represented a reduction of AMD computers.

Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

Directory of Open Access Journals (Sweden)

Sushmita Poddar

2014-06-01

Full Text Available To date the Simian Virus 40 (SV40 is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

Photoionization and Recombination

Science.gov (United States)

Nahar, Sultana N.

2000-01-01

Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

Successful development of recombinant DNA-derived pharmaceuticals.

Science.gov (United States)

Werner, R G; Pommer, C H

1990-11-01

Successful development of recombinant DNA-derived pharmaceuticals, a new class of therapeutic agents, is determined by a variety of factors affecting the selection and positioning of the compound under development. For an efficient development it is of utmost importance that the mechanism of action of the compound selected be understood on a molecular level. The compound's potential therapeutical profile and a strong patent position are key positioning considerations, as well as vital elements in shortening the development phase and protecting innovation. Installation of an interdisciplinary project management team, along with a clear definition of team members' responsibilities, is required to avoid delays and improve communication during development. Selection of the organism to be used in production must take into consideration both the structure of the protein and the quality and safety of the final product. New technologies require a considerable investment in new manufacturing facilities and equipment. Often, the decision for such an investment must be made early and with a high degree of uncertainty. Desired product yield, expected dosage, and estimated market potential are the most important considerations in this decision. Following public disclosure of the plan to develop recombinant DNA-derived products, approval of the production plant and expansion or adaptation to the new process and technology may be delayed. For this reason, they should be considered as a critical step in the overall development phase. Recruitment of qualified staff is a time-consuming and critical element of the production process. Its impact on the product timeline should not be underestimated, especially if such technologies are new to the company. The entire production process must be validated in respect to identity, purity, and safety of the product to guarantee constant product quality, as well as for safety aspects in the environment. Adequate in-process and final product

Gene transfer to the cerebellum.

Science.gov (United States)

Louboutin, Jean-Pierre; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

2010-12-01

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Deriving the coronal hole electron temperature: electron density dependent ionization / recombination considerations

International Nuclear Information System (INIS)

Doyle, John Gerard; Perez-Suarez, David; Singh, Avninda; Chapman, Steven; Bryans, Paul; Summers, Hugh; Savin, Daniel Wolf

2010-01-01

Comparison of appropriate theoretically derived line ratios with observational data can yield estimates of a plasma's physical parameters, such as electron density or temperature. The usual practice in the calculation of the line ratio is the assumption of excitation by electrons/protons followed by radiative decay. Furthermore, it is normal to use the so-called coronal approximation, i.e. one only considers ionization and recombination to and from the ground-state. A more accurate treatment is to include ionization/recombination to and from metastable levels. Here, we apply this to two lines from adjacent ionization stages, Mg IX 368 A and Mg X 625 A, which has been shown to be a very useful temperature diagnostic. At densities typical of coronal hole conditions, the difference between the electron temperature derived assuming the zero density limit compared with the electron density dependent ionization/recombination is small. This, however, is not the case for flares where the electron density is orders of magnitude larger. The derived temperature for the coronal hole at solar maximum is around 1.04 MK compared to just below 0.82 MK at solar minimum.

Structure and evaluation of antibacterial and antitubercular properties of new basic and heterocyclic 3-formylrifamycin SV derivatives obtained via 'click chemistry' approach.

Science.gov (United States)

Pyta, Krystian; Klich, Katarzyna; Domagalska, Joanna; Przybylski, Piotr

2014-09-12

Thirty four novel derivatives of 3-formylrifamycin SV were synthesized via reductive alkylation and copper(I)-catalysed azide-alkyne cycloaddition. According to the obtained results, 'click chemistry' can be successfully applied for modification of structurally complex antibiotics such as rifamycins, with the formation of desired 1,2,3-triazole products. However, when azide-alkyne cycloaddition on 3-formylrifamycin SV derivatives demanded higher amount of catalyst, lower temperature and longer reaction time because of the high volatility of substrates, an unexpected intramolecular condensation with the formation of 3,4-dihydrobenzo[g]quinazoline heterocyclic system took place. Structures of new derivatives in solution were determined using one- and two-dimensional NMR methods and FT-IR spectroscopy. Computational DFT and PM6 methods were employed to correlate their conformation and acid-base properties to biological activity and establish SAR of the novel compounds. Microbiological, physico-chemical (logP, solubility) and structural studies of newly synthesised rifamycins indicated that for the presence of relatively high antibacterial (MIC ~0.01 nmol/mL) and antitubercular (MIC ~0.006 nmol/mL) activities, a rigid and basic substituent at C(3) arm, containing a protonated nitrogen atom "open" toward intermolecular interactions, is required. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

ER Operations Installation of Three FLUTe Soil-Vapor Monitoring Wells (MWL-SV03 MWL-SV04 and MWL-SV05) at the Mixed Waste Landfill.

Energy Technology Data Exchange (ETDEWEB)

Copland, John Robin [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

2014-09-01

This installation report describes the May through July 2014 drilling activities performed for the installation of three multi-port soil-vapor monitoring wells (MWL-SV03, MWL-SV04, and MWL-SV05) at the Mixed Waste Landfill (MWL), which is located at Sandia National Laboratories, New Mexico (SNL/NM). SNL/NM is managed and operated by Sandia Corporation (Sandia), a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy (DOE)/National Nuclear Security Administration. The MWL is designated as Solid Waste Management Unit (SWMU) 76 and is located in Technical Area (TA) III (Figure 1-1). The locations of the three soil-vapor monitoring wells (MWL-SV03, MWL-SV04, and MWL-SV05) are shown in Figure 1-2

Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells

Directory of Open Access Journals (Sweden)

Adcock Ian M

2002-11-01

Full Text Available Abstract Background Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs, the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level. Result DNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2. Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, ΔΨm, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%. 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced ΔΨm increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of ΔΨm, DNA fragmentation was reduced 2 h after exposure to H2O2. Conclusion The data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

Herpes simplex virus type 1-derived recombinant and amplicon vectors.

Science.gov (United States)

Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

2011-01-01

Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

Cryo-electron microscopy of vitrified SV40 minichromosomes: the liquid drop model.

Science.gov (United States)

Dubochet, J; Adrian, M; Schultz, P; Oudet, P

1986-03-01

The structure of SV40 minichromosomes has been studied by cryo-electron microscopy of vitrified thin layers of solution. In high-salt buffer (130 mM NaCl), freshly prepared minichromosomes are condensed into globules 30 nm or more in diameter. On the micrograph, they appear to be formed by the close packing of 10 nm granules which give rise to a 10 nm reflection in the optical diffractogram. The globules can adopt many different conformations. At high concentration, they fuse into a homogeneous 'sea' of closely packed 10 nm granules. In low-salt buffer (less than 10 mM NaCl), the globules open, first into 10 nm filaments, and then into nucleosome-strings. The 'liquid drop' model is proposed to explain the condensed structure of the minichromosome in high-salt buffer: nucleosomes stack specifically on top of one another, thus forming the 10 nm filaments. 10 nm filaments in turn, tend to aggregate laterally. Optimizing both these interactions results in the condensation of 10 nm filaments or portions thereof into a structure similar to that of a liquid. Some implications of this model for the structure of cellular chromatin are discussed.

Replication of simian virus 40 in simian virus 40-transformed hamster kidney cells induced by mitomycin C or 60Co γ irradiation

International Nuclear Information System (INIS)

Rakusanova, T.; Smales, W.P.; Kaplan, J.C.; Black, P.H.

1978-01-01

Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with 60 Co γ irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluorescence techniques, respectively. Only some cells in induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or 60 Co γ irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of γ irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication

One-step simultaneous detection of Ureaplasma parvum and genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology.

Science.gov (United States)

Payne, M S; Furfaro, L L; Tucker, R; Tan, L Y; Mokany, E

2017-08-01

Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10 -5  ng DNA (SV3) and 0.4 × 10 -6  ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. © 2017 The Authors. Letters in Applied Microbiology published by John

Commentaar op art. 563 Sv

NARCIS (Netherlands)

Knoops, G.G.J.; van Laanen, F.; Melai, A.L.; Groenhuijsen, M.S.

2004-01-01

Schrijvers verschaffen een wetenschappelijk commentaar op art. 563 Sv, betreffende de tenuitvoerlegging van andere straffen dan de bij art. 562 Sv bedoelde straffen ingeval van een veroordeelde wiens geestvermogens ziekelijk zijn gestoord.

Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

International Nuclear Information System (INIS)

Tsujimura, T.; Maher, V.M.; McCormick, J.J.; Godwin, A.R.; Liskay, R.M.

1990-01-01

To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

Science.gov (United States)

Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

2013-08-01

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

p53 levels, cell cycle kinetics and radiosensitivity in two SV40 transformed Wi38VA13 fibroblast strains

International Nuclear Information System (INIS)

Werner, F.; Zoelzer, F.; Streffer, C.

2001-01-01

Background: The tumor suppressor protein p53 which can mediate an ionizing radiation-induced G 1 arrest in mammalian cells, forms complexes with SV40 large T antigen (l-T-Ag). We have analyzed the p53 levels, the capability to undergo a G 1 arrest and the radiosensitivity of two SV40 transformed fibroblast strains differing in their large T antigen expression. Material and Methods: One of the two strains (VA13F) is the commercially available form of Wi38VA13, the other (VA13E) arose spontaneously from the original one in our laboratory. Their p53 levels were measured by means of flow cytometry (FCM) and Western blot (WB) with two p53 antibodies (Ab-3, clone PAb240; Ab-6, clone DO-1; both Oncogene Science). Cell cycle distributions were determined flow cytometrically after BrdU labeling at regular time intervals after exposure to 250 kV X-rays. Radiosensitivity was assessed in a clonogenicity assay. Results: The p53 levels of the two strains corresponded to their large T antigen expression, presumably due to complex formation between the two proteins. The strain with a high p53 level did not show a G 1 arrest and had a relatively high radiosensitivity, whereas the strain with a low p53 level showed a significant G 1 arrest and a lower radiosensitivity. Conclusion: These results suggest that 1. complex formation between the large T antigen and p53 reduces the latter's functionality; 2. in these two strains the G 1 arrest is one of the factors determining radiosensitivity. (orig.) [de

Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

Science.gov (United States)

Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida

2013-01-01

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax. PMID:23863502

Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis.

Science.gov (United States)

Agrawal, L; Louboutin, J-P; Reyes, B A S; Van Bockstaele, E J; Strayer, D S

2006-12-01

Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. As they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells and primary fetal neurons were transduced with rSV40 vectors carrying human SOD1 and/or GPx1 cDNAs, then exposed to gp120. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Transduction efficiency of both neuron populations was >95%, as assayed by immunostaining. Transgene expression was also ascertained by Western blotting and direct assays of enzyme activity. Gp120 induced apoptosis in a high percentage of unprotected NT2-N. Transduction with SV(SOD1) and SV(GPx1) before gp120 challenge reduced neuronal apoptosis by >90%. Even greater protection was seen in cells treated with both vectors in sequence. Given singly or in combination, they protect neuronal cells from HIV-1-gp120 induced apoptosis. We tested whether rSV40 s can deliver antioxidant enzymes to the CNS in vivo: intracerebral injection of SV(SOD1) or SV(GPx1) into the caudate putamen of rat brain yielded excellent transgene expression in neurons. In vivo transduction using SV(SOD1) also protected neurons from subsequent gp120-induced apoptosis after injection of both into the caudate putamen of rat brain. Thus, SOD1 and GPx1 can be delivered by SV40 vectors in vitro or in vivo. This approach may merit consideration for

SvSXP: a Strongylus vulgaris antigen with potential for prepatent diagnosis.

Science.gov (United States)

Andersen, Ulla V; Howe, Daniel K; Dangoudoubiyam, Sriveny; Toft, Nils; Reinemeyer, Craig R; Lyons, Eugene T; Olsen, Susanne N; Monrad, Jesper; Nejsum, Peter; Nielsen, Martin K

2013-04-04

Strongyle parasites are ubiquitous in grazing horses. Strongylus vulgaris, the most pathogenic of the large strongyles, is known for its extensive migration in the mesenteric arterial system. The lifecycle of S. vulgaris is characterised by a long prepatent period where the migrating larvae are virtually undetectable as there currently is no test available for diagnosing prepatent S. vulgaris infection. Presence of S. vulgaris larvae in the arterial system causes endarteritis and thrombosis with a risk of non-strangulating intestinal infarctions. Emergence of anthelmintic resistance among cyathostomins has led to recommendations of reduced treatment intensity by targeting horses that exceed a predetermined strongyle faecal egg count threshold. One study suggests an apparent increase in prevalence of S. vulgaris on farms where reduced anthelmintic treatment intensity has been implemented. These issues highlight the need for an accurate and reliable assay for diagnosing prepatent S. vulgaris infection. Immunoscreening of a larval S. vulgaris cDNA library using hyperimmune serum raised against S. vulgaris excretory/secretory antigens was performed to identify potential diagnostic antigens. Immunoreactive clones were sequenced, one potential antigen was characterised, expressed as a recombinant protein, initially evaluated by western blot (WB) analysis, the diagnostic potential of the IgG subclasses was evaluated by ELISA, and the diagnostic accuracy evaluated using serum from 102 horses with known S. vulgaris infection status. The clone expressing the potential antigen encoded a S. vulgaris SXP/RAL2 homologue. The recombinant protein, rSvSXP, was shown to be a potential diagnostic antigen by WB analysis, and a target of serum IgGa, IgG(T) and total IgG in naturally infected horses, with IgG(T) antibodies being the most reliable indicator of S. vulgaris infection in horses. Evaluation of diagnostic accuracy of the ELISA resulted in a sensitivity of 73.3%, a specificity

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

Science.gov (United States)

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Internal 40K radiation dose to Indians

International Nuclear Information System (INIS)

Ranganathan, S.; Someswara Rao, M.; Nagaratnam, A.; Mishra, U.C.

2002-01-01

A group of 350 Indians from both sexes (7-65 years) representing different regions of India was studied for internal 40 K radiation dose from the naturally occurring body 40 K, which was measured in the National Institute of Nutrition (NIN) whole-body counter. Although the 40 K radioactivity reached a peak value by 18 years in female (2,412 Bq) and by 20 years in male (3,058 Bq) and then varied inversely with age in both sexes, the radiation dose did not show such a trend. Boys and girls of 11 years had annual effective dose of nearly 185 mSv, which decreased during adolescence (165 mSv), increased to 175 mSv by 18-20 years in adults and decreased progressively on further ageing to 99 mSv in males and 69 mSv in females at 65 years. The observed annual effective dose (175 mSv) of the young adults was close to that of the ICRP Reference Man (176 mSv) and Indian Reference Man (175 mSv). With a mean specific activity of 55 Bq/kg for the subjects and a conversion coefficient close to 3 mSv per annum per Bq/kg, the average annual effective dose from the internal 40 K turned out to be 165 mSv for Indians. (author)

The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

Science.gov (United States)

Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

2016-06-01

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

Recombinant-derived chicken growth hormone used for radioimmunoassay

International Nuclear Information System (INIS)

Proudman, J.A.

1984-01-01

The use of recombinant-derived chicken growth hormone (rcGH) in an avian growth hormone (GH) radioimmunoassay (RIA) procedure is described. Antiserum to turkey GH bound 125 I-labeled rcGH, and unlabeled rcGH or turkey GH displaced binding in a dose-related manner. The dose-response curves of sera and pituitary extract from chickens and turkeys were parallel to the rcGH standard curve. Sera from hypophysectomized (hypox) chickens and turkeys produced no dose-response and did not inhibit binding of labeled rcGH. Recovery of rcGH added to hypox sera was quantitative. Modification of the homologous turkey GH RIA protocol of Proudman and Wentworth (1) to use rcGH made possible either an increase in assay sensitivity or a 3-day reduction in incubation time

Repeated Administration of D-Amphetamine Induces Distinct Alterations in Behavior and Metabolite Levels in 129Sv and Bl6 Mouse Strains

Directory of Open Access Journals (Sweden)

Taavi Vanaveski

2018-06-01

Full Text Available The main goal of the study was to characterize the behavioral and metabolomic profiles of repeated administration (for 11 days of d-amphetamine (AMPH, 3 mg/kg i. p., indirect agonist of dopamine (DA, in widely used 129S6/SvEvTac (129Sv and C57BL/6NTac (Bl6 mouse strains. Acute administration of AMPH (acute AMPH induced significantly stronger motor stimulation in Bl6. However, repeated administration of AMPH (repeated AMPH caused stronger motor sensitization in 129Sv compared acute AMPH. Body weight of 129Sv was reduced after repeated saline and AMPH, whereas no change occurred in Bl6. In the metabolomic study, acute AMPH induced an elevation of isoleucine and leucine, branched chain amino acids (BCAA, whereas the level of hexoses was reduced in Bl6. Both BCAAs and hexoses remained on level of acute AMPH after repeated AMPH in Bl6. Three biogenic amines [asymmetric dimethylarginine (ADMA, alpha-aminoadipic acid (alpha-AAA, kynurenine] were significantly reduced after repeated AMPH. Acute AMPH caused in 129Sv a significant reduction of valine, lysophosphatidylcholines (lysoPC a C16:0, lysoPC a C18:2, lysoPC a C20:4, phosphatidylcholine (PC diacyls (PC aa C34:2, PC aa C36:2, PC aa C36:3, PC aa C36:4 and alkyl-acyls (PC ae C38:4, PC ae C40:4. However, repeated AMPH increased the levels of valine and isoleucine, long-chain acylcarnitines (C14, C14:1-OH, C16, C18:1, PC diacyls (PC aa C38:4, PC aa C38:6, PC aa C42:6, PC acyl-alkyls (PC ae C38:4, PC ae C40:4, PC ae C40:5, PC ae C40:6, PC ae C42:1, PC ae C42:3 and sphingolipids [SM(OHC22:1, SM C24:0] compared to acute AMPH in 129Sv. Hexoses and kynurenine were reduced after repeated AMPH compared to saline in 129Sv. The established changes probably reflect a shift in energy metabolism toward lipid molecules in 129Sv because of reduced level of hexoses. Pooled data from both strains showed that the elevation of isoleucine and leucine was a prominent biomarker of AMPH-induced behavioral sensitization

Study of recombinant proteins derived from Ser-2 gene of Bombyx mori

OpenAIRE

STAŠKOVÁ, Tereza

2012-01-01

Four different variants of recombinant proteins derived from Bombyx mori Ser-2 gene were expressed in bacteria. The ability of these proteins to coat hydrofobic surfaces and to support growth of various types of adherent cells in vitro were examined. It was shown that these proteins support cell adhesion and proliferation, and could be used as coating agents to realize surfaces suitable for growth of vertebrate and insect cells.

In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences

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Takashi Irie

2012-01-01

Full Text Available The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.

40 CFR 721.1820 - Bisphenol derivative.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Bisphenol derivative. 721.1820 Section... Substances § 721.1820 Bisphenol derivative. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as bisphenol derivative (PMN No. P-92-509) is...

Simian virus 40 infection in humans and association with human diseases: results and hypotheses

International Nuclear Information System (INIS)

Barbanti-Brodano, Giuseppe; Sabbioni, Silvia; Martini, Fernanda; Negrini, Massimo; Corallini, Alfredo; Tognon, Mauro

2004-01-01

Simian virus 40 (SV40) is a monkey virus that was introduced in the human population by contaminated poliovaccines, produced in SV40-infected monkey cells, between 1955 and 1963. Epidemiological evidence now suggests that SV40 may be contagiously transmitted in humans by horizontal infection, independent of the earlier administration of SV40-contaminated poliovaccines. This evidence includes detection of SV40 DNA sequences in human tissues and of SV40 antibodies in human sera, as well as rescue of infectious SV40 from a human tumor. Detection of SV40 DNA sequences in blood and sperm and of SV40 virions in sewage points to the hematic, sexual, and orofecal routes as means of virus transmission in humans. The site of latent infection in humans is not known, but the presence of SV40 in urine suggests the kidney as a possible site of latency, as it occurs in the natural monkey host. SV40 in humans is associated with inflammatory kidney diseases and with specific tumor types: mesothelioma, lymphoma, brain, and bone. These human tumors correspond to the neoplasms that are induced by SV40 experimental inoculation in rodents and by generation of transgenic mice with the SV40 early region gene directed by its own early promoter-enhancer. The mechanisms of SV40 tumorigenesis in humans are related to the properties of the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag). Tag acts mainly by blocking the functions of p53 and RB tumor suppressor proteins, as well as by inducing chromosomal aberrations in the host cell. These chromosome alterations may hit genes important in oncogenesis and generate genetic instability in tumor cells. The clastogenic activity of Tag, which fixes the chromosome damage in the infected cells, may explain the low viral load in SV40-positive human tumors and the observation that Tag is expressed only in a fraction of tumor cells. 'Hit and run' seems the most plausible mechanism to support this situation. The small tag

p53-stabilizing Agent CP-31398 Prevents Growth and Invasion of Urothelial Cancer of the Bladder in Transgenic UPII-SV40T Mice

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Venkateshwar Madka

2013-08-01

Full Text Available The high prevalence of bladder cancer and its recurrence make it an important target for chemoprevention. About half of invasive urothelial tumors have mutations in p53. We determined the chemopreventive efficacy of a p53-stabilizing agent, CP-31398, in a transgenic UPII-SV40T mouse model of bladder transitional cell carcinoma (TCC that strongly resembles human TCC. After genotyping, six-week-old UPII-SV40T mice (n = 30/group were fed control (AIN-76A or experimental diets containing 150 or 300 ppm of CP-31398 for 34 weeks. Progression of bladder cancer growth was monitored by magnetic resonance imaging. At 40 weeks of age, all mice were killed; urinary bladders were collected to determine weights, tumor incidence, and histopathology. There was a significant increase in bladder weights of transgenic versus wild-type mice (male: 140.2 mg vs 27.3 mg, P < .0001; female: 34.2 mg vs 14.8 mg, P < .0001. A significant decrease in the bladder tumor weights (by 68.6–80.2%, P < .0001 in males and by 36.9–55.3%, P < .0001 in females was observed in CP-31398-treated mice. Invasive papillary TCC incidence was 100% in transgenic mice fed control diet. Both male and female mice exposed to CP-31398 showed inhibition of invasive TCC. CP-31398 (300 ppm completely blocked invasion in female mice. Molecular analysis of the bladder tumors showed an increase in apoptosis markers (p53, p21, Bax, and Annexin V with a decrease in vascular endothelial growth factor in transgenic mice fed CP-31398. These results suggest that p53-modulating agents can serve as potential chemopreventive agents for bladder TCC.

Oltar Sv. Jeronima u crkvi Sv. Šime u Zadru i radionica Bettamelli

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Bojan Goja

2012-12-01

Full Text Available U radu se na temelju arhivskih podataka govori o oltaru Sv. Jeronima u crkvi Sv. Šime u Zadru. Od ranije se zna da je oltar dala podići i o njegovu se uređenju brinula bratovština hrvatskih i albanskih vojnika (Croatti a cavallo i Soldati Albanesi u službi Mletačke Republike, osnovana 1675. godine u Zadru. Novim je arhivskim istraživanjima utvrđeno da je dana 26. rujna 1694. godine bratovština odobrila izdatak u iznosu od 200 srebrnih dukata, namijenjenih venecijanskim klesarima braći Bettamelli kao predujam za izradu oltara te da su radovi na njegovu podizanju započeti u travnju 1696. godine. Na temelju zapisa o gradnji oltara iznijeta je pretpostavka da se grb na istočnom pilastru stipesa oltara, ranije povezivan s predstavnicima obitelji Civran, odnosi na zadarskog plemića i istaknutog zapovjednika u mletačkoj vojsci, Šimuna Fanfognu (Zadar, 7. travnja 1663. - Lendinara, 6. ožujka 1707. koji je obavljao i dužnost prokuratora oltara. Brojni radovi na uređenju oltara i kapele Sv. Jeronima obavljani su i tijekom čitavog 18. stoljeća, a na njima su bili uposleni brojni mjesni majstori različitih struka: graditelji Antonio Piovesana (1742. i Antonio Bernardini (1789., oltarist Girolamo Picco (1756., marangon Domenico Tomaselli (1743., kovač Antonelli (1744. te zlatari Zorzi Cullisich (1738., Nicolò Giurovich (1752. i Giuseppe Rado (1755.. Donose se još neke zanimljivosti vezane uz uređenje oltara i djelovanje bratovštine Sv. Jeronima.

Svømmehaller og krav til energieffektivitet

OpenAIRE

Øen, Martin Nerhus

2010-01-01

Svømmehaller er en spesiell bygningstype, som med høy innendørs temperatur og luftfuktighet, skiller seg fra andre bygninger som boliger og kontorbygg. Den høye innetemperaturen, forbruket av varmtvann, og fordampning fra svømmebassenget fører til et stort energiforbruk. Svømmehaller er ikke gitt egne krav i Teknisk forskrift til plan- og bygningsloven (TEK), til tross for at energiforbruket kan være tre ganger høyere enn kravene gitt for idrettshaller. Målet med denne rapporten er å finne fo...

Levetiracetam reverses synaptic deficits produced by overexpression of SV2A.

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Amy Nowack

Full Text Available Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a ∼1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions.

Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

International Nuclear Information System (INIS)

Aravalli, Rajagopal N.; Behnan Sahin, M.; Cressman, Erik N.K.; Steer, Clifford J.

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Establishment and characterization of a unique 1 {mu}m diameter liver-derived progenitor cell line

Energy Technology Data Exchange (ETDEWEB)

Aravalli, Rajagopal N., E-mail: arava001@umn.edu [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Behnan Sahin, M. [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Cressman, Erik N.K. [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 {mu}m in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.

Science.gov (United States)

Sevillano, Alejandro M; Fernández-Borges, Natalia; Younas, Neelam; Wang, Fei; R Elezgarai, Saioa; Bravo, Susana; Vázquez-Fernández, Ester; Rosa, Isaac; Eraña, Hasier; Gil, David; Veiga, Sonia; Vidal, Enric; Erickson-Beltran, Melissa L; Guitián, Esteban; Silva, Christopher J; Nonno, Romolo; Ma, Jiyan; Castilla, Joaquín; R Requena, Jesús

2018-01-01

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

Cell and molecular biology of simian virus 40: implications for human infections and disease

Science.gov (United States)

Butel, J. S.; Lednicky, J. A.

1999-01-01

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology.

Mesothelioma mortality in Europe: impact of asbestos consumption and simian virus 40

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Rehak Peter

2006-11-01

Full Text Available Abstract Background It is well established that asbestos is the most important cause of mesothelioma. The role of simian virus 40 (SV40 in mesothelioma development, on the other hand, remains controversial. This potential human oncogene has been introduced into various populations through contaminated polio vaccines. The aim of this study was to investigate whether the possible presence of SV40 in various European countries, as indicated either by molecular genetic evidence or previous exposure to SV40-contaminated vaccines, had any effect on pleural cancer rates in the respective countries. Methods We conducted a Medline search that covered the period from January 1969 to August 2005 for reports on the detection of SV40 DNA in human tissue samples. In addition, we collected all available information about the types of polio vaccines that had been used in these European countries and their SV40 contamination status. Results Our ecological analysis confirms that pleural cancer mortality in males, but not in females, correlates with the extent of asbestos exposure 25 – 30 years earlier. In contrast, neither the presence of SV40 DNA in tumor samples nor a previous vaccination exposure had any detectable influence on the cancer mortality rate in neither in males (asbestos-corrected rates nor in females. Conclusion Using the currently existing data on SV40 prevalence, no association between SV40 prevalence and asbestos-corrected male pleural cancer can be demonstrated.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

International Nuclear Information System (INIS)

Tátrai, Péter; Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Buchan, Gyöngyi; Mádi, András; Uher, Ferenc

2012-01-01

Highlights: ► We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. ► hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. ► SV40T introduced along with hTERT abrogates proliferation control and multipotency. ► hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC hTERT , ASC Bmi-1 , ASC Bmi-1+hTERT and ASC SV40T+hTERT were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC Bmi-1 had limited replicative potential, while the rapidly proliferating ASC SV40T+hTERT acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC hTERT and ASC hTERT+Bmi-1 , on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC hTERT also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC hTERT are prone to transformation during extensive

Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

Science.gov (United States)

Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers. Copyright 2009 Elsevier Inc. All rights reserved.

Therapeutic Recombinant Monoclonal Antibodies

Science.gov (United States)

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

Energy Technology Data Exchange (ETDEWEB)

Gong, Y.; Li, X.M.; Shapiro, L.J. [UCSF School of Medicine, San Francisco, CA (United States)] [and others

1994-09-01

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand, and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

Science.gov (United States)

Pliaka, V; Kyriakopoulou, Z; Tsakogiannis, D; Ruether, I G A; Gartzonika, C; Levidiotou-Stefanou, S; Krikelis, A; Markoulatos, P

2010-12-01

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

Directory of Open Access Journals (Sweden)

Young Hee Joung

2016-10-01

Full Text Available Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO-recommended vaccines including hepatitis B (HepB. HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV, however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed.

Construction and expression of a recombinant antibody-targeted plasminogen activator

International Nuclear Information System (INIS)

Schnee, J.M.; Runge, M.S.; Matsueda, G.A.; Hudson, N.W.; Seidman, J.G.; Haber, E.; Quertermous, T.

1987-01-01

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

SV-AUTOPILOT: optimized, automated construction of structural variation discovery and benchmarking pipelines.

Science.gov (United States)

Leung, Wai Yi; Marschall, Tobias; Paudel, Yogesh; Falquet, Laurent; Mei, Hailiang; Schönhuth, Alexander; Maoz Moss, Tiffanie Yael

2015-03-25

Many tools exist to predict structural variants (SVs), utilizing a variety of algorithms. However, they have largely been developed and tested on human germline or somatic (e.g. cancer) variation. It seems appropriate to exploit this wealth of technology available for humans also for other species. Objectives of this work included: a) Creating an automated, standardized pipeline for SV prediction. b) Identifying the best tool(s) for SV prediction through benchmarking. c) Providing a statistically sound method for merging SV calls. The SV-AUTOPILOT meta-tool platform is an automated pipeline for standardization of SV prediction and SV tool development in paired-end next-generation sequencing (NGS) analysis. SV-AUTOPILOT comes in the form of a virtual machine, which includes all datasets, tools and algorithms presented here. The virtual machine easily allows one to add, replace and update genomes, SV callers and post-processing routines and therefore provides an easy, out-of-the-box environment for complex SV discovery tasks. SV-AUTOPILOT was used to make a direct comparison between 7 popular SV tools on the Arabidopsis thaliana genome using the Landsberg (Ler) ecotype as a standardized dataset. Recall and precision measurements suggest that Pindel and Clever were the most adaptable to this dataset across all size ranges while Delly performed well for SVs larger than 250 nucleotides. A novel, statistically-sound merging process, which can control the false discovery rate, reduced the false positive rate on the Arabidopsis benchmark dataset used here by >60%. SV-AUTOPILOT provides a meta-tool platform for future SV tool development and the benchmarking of tools on other genomes using a standardized pipeline. It optimizes detection of SVs in non-human genomes using statistically robust merging. The benchmarking in this study has demonstrated the power of 7 different SV tools for analyzing different size classes and types of structural variants. The optional merge

Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

Science.gov (United States)

Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

2015-01-01

Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

Energy Technology Data Exchange (ETDEWEB)

Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

2012-05-25

Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

Association between simian virus 40 and non-Hodgkin lymphoma

Science.gov (United States)

Vilchez, Regis A.; Madden, Charles R.; Kozinetz, Claudia A.; Halvorson, Steven J.; White, Zoe S.; Jorgensen, Jeffrey L.; Finch, Chris J.; Butel, Janet S.

2002-01-01

BACKGROUND: Non-Hodgkin lymphoma has increased in frequency over the past 30 years, and is a common cancer in HIV-1-infected patients. Although no definite risk factors have emerged, a viral cause has been postulated. Polyomaviruses are known to infect human beings and to induce tumours in laboratory animals. We aimed to identify which one of the three polyomaviruses able to infect human beings (simian virus 40 [SV40], JC virus, and BK virus) was associated with non-Hodgkin lymphoma. METHODS: We analysed systemic non-Hodgkin lymphoma from 76 HIV-1-infected and 78 HIV-1-uninfected patients, and non-malignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumours; 54 colon and breast carcinoma samples served as cancer controls. We used PCR followed by Southern blot hybridisation and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. FINDINGS: Polyomavirus T antigen sequences, all of which were SV40-specific, were detected in 64 (42%) of 154 non-Hodgkin lymphomas, none of 186 non-malignant lymphoid samples, and none of 54 control cancers. This difference was similar for HIV-1-infected patients and HIV-1-uninfected patients alike. Few tumours were positive for both SV40 and Epstein-Barr virus. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B-cell and follicular-type lymphomas. INTERPRETATION: SV40 is significantly associated with some types of non-Hodgkin lymphoma. These results add lymphomas to the types of human cancers associated with SV40.

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

International Nuclear Information System (INIS)

Debenham, P.G.; Webb, M.B.T.; Masson, W.K.; Cox, R.

1984-01-01

An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10 -5 and 10 -4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication

International Nuclear Information System (INIS)

Scaria, A.; Edenberg, H.J.

1987-01-01

The authors examined the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV40 DNA synthesis in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m 2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m 2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication. (Auth.)

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

Science.gov (United States)

Kukiełka, E; Cederbaum, A I

1995-04-15

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

Replicative intermediates in UV-irradiated Simian virus 40

International Nuclear Information System (INIS)

Clark, J.M.; Hanawalt, P.C.

1984-01-01

The authors have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m 2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [ 3 H]thymidine. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h by which time the size of the newly-synthesized DNA exceeded the interdimer distance. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strand contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from 0V-damaged SV40 replicative intermediates. (Auth.)

Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

Science.gov (United States)

Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

2016-12-30

This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

Science.gov (United States)

Butel, J. S.

2000-01-01

From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

Energy Technology Data Exchange (ETDEWEB)

Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

Somatic mutation and recombination induced with reactor thermal neutrons in Drosophila melanogaster

International Nuclear Information System (INIS)

Zambrano A, F.; Guzman R, J.; Paredes G, L.; Delfin L, A.

1997-01-01

The SMART test of Drosophila melanogaster was used to quantify the effect over the somatic mutation and recombination induced by thermal and fast neutrons at the TRIGA Mark III reactor of the ININ at the power of 300 k W for times of 30, 60 and 120 minutes with total equivalent doses respectively of 20.8, 41.6 and 83.2 Sv. A linear relation between the radiation equivalent dose and the frequency of the genetic effects such as mutation and recombination was observed. The obtained results allow to conclude that SMART is a sensitive system to the induced damage by neutrons, so this can be used for studying its biological effects. (Author)

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Science.gov (United States)

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

2017-07-04

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Specifications for Testing Procedures at Svåheia

DEFF Research Database (Denmark)

Margheritini, Lucia; Kofoed, Jens Peter

This report is realized in order to define testing procedures of the SSG pilot in Svåheia location. This will be done by listing all the relevant parameters related to the structure performance.......This report is realized in order to define testing procedures of the SSG pilot in Svåheia location. This will be done by listing all the relevant parameters related to the structure performance....

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa

DEFF Research Database (Denmark)

Plesner, Bitten; Westh, Peter; Nielsen, Anders D.

2011-01-01

The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated® recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular...... dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature Tm measured by differential scanning calorimetry (DSC) and the temperature of aggregation, Tagg, measured by light scattering (LS......) both increased with GlycoPEGylation. Both Tm and Tagg were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure...

Progressive paralysis associated with diffuse astrocyte anaplasia in delta 202 mice homozygous for a transgene encoding the SV40 T antigen.

Science.gov (United States)

López-Revilla, Rubén; Soto-Zárate, Carlos; Ridaura, Cecilia; Chávez-Dueñas, Lucía; Paul, Dieter

2004-03-01

A convenient transgenic astrocytoma model in delta202 mice, homozygous for a construct encoding the early region of the SV40 virus genome, is described. In the offspring of crosses between delta202 mice heterozygous for the transgene nearly 60% were transgenic; one third of these developed progressive paralysis starting in the hindlimbs at approximately 35 days of age and died at 90 +/- 30 days of age. In affected mice proliferating-non-neuronal cells immunostained with antibodies to the GFAP, an astrocyte marker, whose number increased with age were found in the white matter of the brain, cerebellum and spinal cord, and progressive degeneration and necrosis of spinal motoneurons was observed that-may explain the paralysis. The early onset and reproducible time course of the neurological disease suggest that homozygous delta202 mice, whose proliferating astrocytes appear to damage spinal motoneurons, are a useful model to study astrocyte differentiation, function and tumorigenesis.

Development and validation of a catalytic recombiner model for the containment code RALOC MOD4.0

International Nuclear Information System (INIS)

Rohde, J.; Klein-Hebling, W.; Chakraborty, A.K.

1997-01-01

This paper reports on the development of a catalytic recombiner model for the containment code RALOC MOD4.0 /KLH 95, KLH 96/ and the detailed validation work, carried out at GRS. The model was qualified by using the results of medium and large scale experiments, being performed in Germany /KAN 91/. The comparison of measured data with the calculations demonstrates, that this new model is suitable for real plant applications to investigate the overall effectiveness of a catalytic recombiner system under severe accident conditions for large dry containments of German PWR design. The results of such investigations will serve as the basis to work out some guidance for the determination of the system capacity needed and an optimal positioning of such devices in containments. (author)

Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

Science.gov (United States)

Amirhaeri, S; Wohlrab, F; Wells, R D

1995-02-17

The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

Electron-ion recombination rates for merged-beams experiments

International Nuclear Information System (INIS)

Pajek, M.

1994-01-01

Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

Radiation doses deriving from patients undergoing 111In-DTPA-d-Phe-1-octreotide scintigraphy

International Nuclear Information System (INIS)

Kurtaran, A.; Pfreitfellner, J.; Smith-Jones, P.; Schaffarich, P.; Niederle, B.; Raderer, M.; Virgolini, I.; Bergmann, H.; Havlik, E.

1997-01-01

The purpose of this study was to estimate the radiation doses to nursing staff, other patients, accompanying persons and family members deriving from patients undergoing 111 In-DTPA-d-Phe-1-octreotide ( 111 In-OCT) scintigraphy. Dose rates were measured from 16 patients who had received an intravenous injection of 140±40 MBq 111 In-OCT. The measurements were performed at three different distances (0.5, 1 and 2 m) at 10-20 min, 5-7 h and 24 h (and in some cases, up to 48 h) after administration of 111 In-OCT. The effective half-lives of the biexponential decrease of the dose rates were estimated to be 2.94±0.27 h (T 1 ) and 65.17±0.58 h (T 2 ). The calculated maximum dose to other persons in the waiting area was 27.2 μSv, to family members 61.5 μSv, to nursing staff in a ward 24.1 μSv and to neighbouring patients in the ward 69.5 μSv. Our results clearly demonstrate that the calculated maximum radiation exposure to accompanying persons, personnel, family members and other patients is well below the maximum annual dose limit for non-professionally exposed persons. (orig.)

A novel splice variant of supervillin, SV5, promotes carcinoma cell proliferation and cell migration

International Nuclear Information System (INIS)

Chen, Xueran; Yang, Haoran; Zhang, Shangrong; Wang, Zhen; Ye, Fang; Liang, Chaozhao; Wang, Hongzhi; Fang, Zhiyou

2017-01-01

Supervillin is an actin-associated protein that regulates actin dynamics by interacting with Myosin II, F-actin, and Cortactin to promote cell contractility and cell motility. Two splicing variants of human Supervillin (SV1 and SV4) have been reported in non-muscle cells; SV1 lacks 3 exons present in the larger isoform SV4. SV2, also called archvillin, is present in striated muscle; SV3, also called smooth muscle archvillin or SmAV, was cloned from smooth muscle. In the present study, we identify a novel splicing variant of Supervillin (SV5). SV5 contains a new splicing pattern. In the mouse tissues and cell lines examined, SV5 was predominantly expressed in skeletal and cardiac muscles and in proliferating cells, but was virtually undetectable in most normal tissues. Using RNAi and rescue experiments, we show here that SV5 displays altered functional properties in cancer cells, and regulates cell proliferation and cell migration.

Comparative human cellular radiosensitivity: Pt. 1

International Nuclear Information System (INIS)

Arlett, C.F.; Green, M.H.L.; Priestley, A.; Harcourt, S.A.; Mayne, L.V.

1988-01-01

The authors compared cell killing following 60 Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. They confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40-virus but immortal cells are more gamma radiation resistant than corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that expression of SV40 T-antigen, rather than immortalization per se is responsible for the change. (author)

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

Energy Technology Data Exchange (ETDEWEB)

Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

2015-10-15

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

International Nuclear Information System (INIS)

Albariño, César G.; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

2015-01-01

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

OpenAIRE

Kukiełka, E; Cederbaum, A I

1995-01-01

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid...

Development of direct reading dosimeters for the dose 0-3 mSv and 0-5 mSv ranges for personnel monitoring

International Nuclear Information System (INIS)

Gaikwad, P.V.; Shirkar, Y.B.; Patil, A.S.; Madgaonkar, P.P.; Kale, K.L.; Guhagarkar, H.V.; Gandhi, D.P.; Gupta, S.K.; Kothiyal, G.P.; Sahni, V.C.

1998-01-01

Direct reading dosimeters (DRDs) are widely used to measure cumulative dose received by personnel working at nuclear reactor sites or in other environment having x- and gamma rays. A DRD operates on the principle of gold leaf electroscope, and is a small, rugged, hermetically sealed, self reading type device easily carried by an individual in his pocket. The development of dosimeters suitable for the dose ranges 0-3 mSv and 0-5 mSv is reported

Recent and historical recombination in the admixed Norwegian Red cattle breed

Directory of Open Access Journals (Sweden)

Grove Harald

2011-01-01

Full Text Available Abstract Background Comparison of recent patterns of recombination derived from linkage maps to historical patterns of recombination from linkage disequilibrium (LD could help identify genomic regions affected by strong artificial selection, appearing as reduced recent recombination. Norwegian Red cattle (NRF make an interesting case study for investigating these patterns as it is an admixed breed with an extensively recorded pedigree. NRF have been under strong artificial selection for traits such as milk and meat production, fertility and health. While measures of LD is also crucial for determining the number of markers required for association mapping studies, estimates of recombination rate can be used to assess quality of genomic assemblies. Results A dataset containing more than 17,000 genome-wide distributed SNPs and 2600 animals was used to assess recombination rates and LD in NRF. Although low LD measured by r2 was observed in NRF relative to some of the breeds from which this breed originates, reports from breeds other than those assessed in this study have described more rapid decline in r2 at short distances than what was found in NRF. Rate of decline in r2 for NRF suggested that to obtain an expected r2 between markers and a causal polymorphism of at least 0.5 for genome-wide association studies, approximately one SNP every 15 kb or a total of 200,000 SNPs would be required. For well known quantitative trait loci (QTLs for milk production traits on Bos Taurus chromosomes 1, 6 and 20, map length based on historic recombination was greater than map length based on recent recombination in NRF. Further, positions for 130 previously unpositioned contigs from assembly of the bovine genome sequence (Btau_4.0 found using comparative sequence analysis were validated by linkage analysis, and 28% of these positions corresponded to extreme values of population recombination rate. Conclusion While LD is reduced in NRF compared to some of the

Transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm Olavius algarvensis.

Science.gov (United States)

Wippler, Juliane; Kleiner, Manuel; Lott, Christian; Gruhl, Alexander; Abraham, Paul E; Giannone, Richard J; Young, Jacque C; Hettich, Robert L; Dubilier, Nicole

2016-11-21

The gutless marine worm Olavius algarvensis has a completely reduced digestive and excretory system, and lives in an obligate nutritional symbiosis with bacterial symbionts. While considerable knowledge has been gained of the symbionts, the host has remained largely unstudied. Here, we generated transcriptomes and proteomes of O. algarvensis to better understand how this annelid worm gains nutrition from its symbionts, how it adapted physiologically to a symbiotic lifestyle, and how its innate immune system recognizes and responds to its symbiotic microbiota. Key adaptations to the symbiosis include (i) the expression of gut-specific digestive enzymes despite the absence of a gut, most likely for the digestion of symbionts in the host's epidermal cells; (ii) a modified hemoglobin that may bind hydrogen sulfide produced by two of the worm's symbionts; and (iii) the expression of a very abundant protein for oxygen storage, hemerythrin, that could provide oxygen to the symbionts and the host under anoxic conditions. Additionally, we identified a large repertoire of proteins involved in interactions between the worm's innate immune system and its symbiotic microbiota, such as peptidoglycan recognition proteins, lectins, fibrinogen-related proteins, Toll and scavenger receptors, and antimicrobial proteins. We show how this worm, over the course of evolutionary time, has modified widely-used proteins and changed their expression patterns in adaptation to its symbiotic lifestyle and describe expressed components of the innate immune system in a marine oligochaete. Our results provide further support for the recent realization that animals have evolved within the context of their associations with microbes and that their adaptive responses to symbiotic microbiota have led to biological innovations.

Insertion of liver enriched transcription factor hepatocyte nuclear factor-4 (HNF-4) in a vector which contains simian virus (SV40) promoter

International Nuclear Information System (INIS)

Al-Nbaheen, M.; Pourzand, C.; Tyrrell, R.M.

2006-01-01

One way of targeting gene expression in vivo is to control transcription using a tissue-specific regulatory system. Tissue specific promoters or enhancers are in use in transgenic animals and could be utilized in medical for gene therapy. At present the usual method for selection of a tissue-specific promoter is to identify a gene, which is expressed at unusually high level in the target tissue, and then to use the promoter for this gene to drive expression of another therapeutic gene in the target tissue. This approach is logical but does not always lead to high levels of gene expression. A second approach is to investigate the scope for discovery of synthetic specific promoters using a target tissue. The objective of the work described in this paper was to use both approach to design plasmid DNA expression vectors that would carry liver-specific promoter/enhancer linked to reporter gene (i.e. luciferase). Then transfect these vectors to both liver-derived and non-liver cell lines. This is followed by evaluation of the liver-specificity of each construct by measuring the basal level expression of the reporter gene (i.e. luciferase activity) in both cell lines. Hepatocyte nuclear factor-4 (HNF-4) is liver-enriched transcription factor used to design new synthetic enhancers by inserting a tandem array of 1', 3' or 5' repeats of the HNF-4 binding site upstream of the SV40 promoter linked to the luciferase reporter gene within an Epstein-Barr virus (EBV)-based vector, p 706. The results of transfection revealed that unexpectedly the HNF-4 binding sites in these constructs act as a repressor rather than enhancer of the liver-specific expression of the luciferase gene. (author)

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

International Nuclear Information System (INIS)

Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

2011-01-01

With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

Initiation of simian virus 40 DNA replication in vitro: Pulse-chase experiments identify the first labeled species as topologically unwound

International Nuclear Information System (INIS)

Bullock, P.A.; Seo, Yeon Soo; Hurwitz, J.

1989-01-01

A distinct unwound form of DNA containing the simian virus 40 (SV40) origin is produced in replication reactions carried out in mixtures containing crude fractions prepared from HeLa cells. This species, termed form U R , comigrates on chloroquine-containing agarose gels with the upper part of the previously described heterogeneous highly unwound circular DNA, form U. As with form U, formation of form U R is dependent upon the SV40 tumor (T) antigen. Pulse-chase experiments demonstrate that the first species to incorporate labeled deoxyribonucleotides comigrates with form U R . Restriction analyses of the products of the pulse-chase experiments show that initiation occurs at the SV40 origin and then proceeds outward in a bidirectional manner. These experiments establish form U R as the earliest detectable substrate for SV40 DNA replication and suggest that SV40 DNA replication initiates on an unwound species

Interface recombination influence on carrier transport

International Nuclear Information System (INIS)

Konin, A

2013-01-01

A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

40K activities and potassium concentrations in tobacco samples of Mexican cigarettes

International Nuclear Information System (INIS)

Martinez, T.; Navarrete, M.; Cabrera, L.; Ramos, A.; Vazquez, K.; Juarez, F.

2007-01-01

Nine brands of tobacco cigarettes manufactured and distributed in the Mexican market were analyzed by γ-spectrometry to certify their nonartificial radioactive contamination. Since natural occurring radioactive materials (NORM) 40 K, 232 Th, 235 U, and 239 U (and decay products from the latter three nuclides) are the main sources for human radiation exposure, the aim of this work was to determine the activity of 40 K and potassium concentration. Averages of 40 K and potassium concentration were of 1.29 ± 0.18 Bq x g -1 , and 4.0 ± 0.57%. The annual dose equivalents to the whole body from ingestion and inhalation of 26 Bq 40 K were 0.23 μSv and 15.8 μSv, respectively. The corresponding 50 years committed dose equivalents was 0.23 μSv. The total committed dose to the lungs due to inhalation of 40 K in tobacco was 16 μSv. Potassium concentrations obtained in this work were in the same range of those obtained by INAA, so showing that the used technique is acute, reproducible, and accessible to laboratories equipped with low background scintillation detectors. (author)

Dielectronic and Trielectronic Recombination Rate Coefficients of Be-like Ar14+

Science.gov (United States)

Huang, Z. K.; Wen, W. Q.; Xu, X.; Mahmood, S.; Wang, S. X.; Wang, H. B.; Dou, L. J.; Khan, N.; Badnell, N. R.; Preval, S. P.; Schippers, S.; Xu, T. H.; Yang, Y.; Yao, K.; Xu, W. Q.; Chuai, X. Y.; Zhu, X. L.; Zhao, D. M.; Mao, L. J.; Ma, X. M.; Li, J.; Mao, R. S.; Yuan, Y. J.; Wu, B.; Sheng, L. N.; Yang, J. C.; Xu, H. S.; Zhu, L. F.; Ma, X.

2018-03-01

Electron–ion recombination of Be-like 40Ar14+ has been measured by employing the electron–ion merged-beams method at the cooler storage ring CSRm. The measured absolute recombination rate coefficients for collision energies from 0 to 60 eV are presented, covering all dielectronic recombination (DR) resonances associated with 2s 2 → 2s2p core transitions. In addition, strong trielectronic recombination (TR) resonances associated with 2s 2 → 2p 2 core transitions were observed. Both DR and TR processes lead to series of peaks in the measured recombination spectrum, which have been identified by the Rydberg formula. Theoretical calculations of recombination rate coefficients were performed using the state-of-the-art multi-configuration Breit–Pauli atomic structure code AUTOSTRUCTURE to compare with the experimental results. The plasma rate coefficients for DR+TR of Ar14+ were deduced from the measured electron–ion recombination rate coefficients in the temperature range from 103 to 107 K, and compared with calculated data from the literature. The experimentally derived plasma rate coefficients are 60% larger and 30% lower than the previously recommended atomic data for the temperature ranges of photoionized plasmas and collisionally ionized plasmas, respectively. However, good agreement was found between experimental results and the calculations by Gu and Colgan et al. The plasma rate coefficients deduced from experiment and calculated by the current AUTOSTRUCTURE code show agreement that is better than 30% from 104 to 107 K. The present results constitute a set of benchmark data for use in astrophysical modeling.

SV2: accurate structural variation genotyping and de novo mutation detection from whole genomes.

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Antaki, Danny; Brandler, William M; Sebat, Jonathan

2018-05-15

Structural variation (SV) detection from short-read whole genome sequencing is error prone, presenting significant challenges for population or family-based studies of disease. Here, we describe SV2, a machine-learning algorithm for genotyping deletions and duplications from paired-end sequencing data. SV2 can rapidly integrate variant calls from multiple structural variant discovery algorithms into a unified call set with high genotyping accuracy and capability to detect de novo mutations. SV2 is freely available on GitHub (https://github.com/dantaki/SV2). jsebat@ucsd.edu. Supplementary data are available at Bioinformatics online.

Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements.

Science.gov (United States)

Spuesens, Emiel B M; van de Kreeke, Nick; Estevão, Silvia; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

2011-02-01

Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

The Use of Recombinant Human Platelet-Derived Growth Factor for Maxillary Sinus Augmentation.

Science.gov (United States)

Kubota, Atsushi; Sarmiento, Hector; Alqahtani, Mohammed Saad; Llobell, Arturo; Fiorellini, Joseph P

The maxillary sinus augmentation procedure has become a predictable treatment to regenerate bone for implant placement. The purpose of this study was to evaluate the effect of recombinant human platelet-derived growth factor BB (rhPDGF-BB) combined with a deproteinized cancellous bovine bone graft for sinus augmentation. The lateral window approach was used for maxillary sinuses with minimal residual bone. After a healing period of 4 months, dental implants were placed and then restored following a 2-month osseointegration period. The result demonstrated increased bone height and ISQ values and a 100% survival rate. This study indicates that the addition of rhPDGF-BB to deproteinized cancellous bovine bone accelerated the healing period in maxillary sinuses with minimal native bone.

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Science.gov (United States)

Weber, Eva; Guth, Christina; Weiss, Ingrid M

2012-01-01

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

Science.gov (United States)

Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

1993-03-01

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Solid state fermentation and production of rifamycin SV using Amycolatopsis mediterranei.

Science.gov (United States)

Nagavalli, M; Ponamgi, S P D; Girijashankar, V; Venkateswar Rao, L

2015-01-01

Production of Rifamycin SV from cheaper agro-industrial by-products using mutant strain of Amycolatopsis mediterranei OVA5-E7 in solid state fermentation (SSF) was optimized. Among the agro-based substrates used, ragi bran was found suitable for maximizing the yield of Rifamycin SV (1310 mg 100 g(-1) ds). The yield can be further enhanced to 19·7 g Kg(-1) of dry substrate by supplementing the substrate with deoiled cotton cake (10% w/w) using optimized fermentation parameters such as maintaining 80% moisture, pH 7·0, 30°C incubation temperature, inoculum 25% v/w and carrying the solid state fermenting for 9 days. Manipulating these seven specifications, the end product yield achieved in our experimentation was 20 g of Rifamycin SV Kg(-1) ds. Eventually, an overall 5-fold improvement in Rifamycin SV production was achieved. Antibiotics such as rifamycin are broad-spectrum antimicrobial drugs used in large-scale worldwide as human medicine towards controlling diseases. Amycolatopsis mediterranei strain which produces this antibiotic was earlier used in submerged fermentation yielded lower amounts of rifamycin. By employing cheaper agro-industrial by-products, we produced upto 20 g rifamycin SV per Kg dry substrate used under optimized solid state fermentation conditions. Keeping in view, the role of rifamycin in meeting the medical demands of world's increasing population; we successfully used an improved strain on cheaper substrates with optimized fermentation parameters and achieved a 5-fold improvement in rifamycin SV production. © 2014 The Society for Applied Microbiology.

Simian virus 40 vectors for pulmonary gene therapy

Directory of Open Access Journals (Sweden)

Oppenheim Ariella

2007-10-01

Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

13C and 15N CP/MAS, 1H-15N SCT CP/MAS and FTIR spectroscopy as tools for qualitative detection of the presence of zwitterionic and non-ionic forms of ansa-macrolide 3-formylrifamycin SV and its derivatives in solid state.

Science.gov (United States)

Przybylski, Piotr; Pyta, Krystian; Klich, Katarzyna; Schilf, Wojciech; Kamieński, Bohdan

2014-01-01

(13)C, (15)N CP/MAS, including (1)H-(13)C and (1)H-(15)N short contact time CP/MAS experiments, and FTIR methods were applied for detailed structural characterization of ansa-macrolides as 3-formylrifamycin SV (1) and its derivatives (2-6) in crystal and in powder forms. Although HPLC chromatograms for 2/CH3 OH and 2/CH3 CCl3 were the same for rifampicin crystals dissolved in respective solvents, the UV-vis data recorded for them were different in 300-375 nm region. Detailed solid state (13)C and (15)N CP/MAS NMR and FTIR studies revealed that rifampicin (2), in contrast to 3-formylrifamycin SV (1) and its amino derivatives (3-6), can occur in pure non-ionic or zwitterionic forms in crystal and in pure these forms or a mixture of them in a powder. Multinuclear CP/MAS and FTIR studies demonstrated also that 3-6 derivatives were present exclusively in pure zwitterionic forms, both in powder and in crystal. On the basis of the solid state NMR and FTIR studies, two conformers of 3-formylrifamycin SV were detected in powder form due to the different orientations of carbonyl group of amide moiety. The PM6 molecular modeling at the semi-empirical level of theory, allowed visualization the most energetically favorable non-ionic and zwitterionic forms of 1-6 antibiotics, strongly stabilized via intramolecular H-bonds. FTIR studies indicated that the originally adopted forms of these type antibiotics in crystal or in powder are stable in standard laboratory conditions in time. The results presented point to the fact that because of a possible presence of two forms of rifampicin (compound 2), quantification of the content of this antibiotic in relevant pharmaceuticals needs caution. Copyright © 2013 John Wiley & Sons, Ltd.

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

International Nuclear Information System (INIS)

Hashimoto, Tomoko; Furuyama, Jun-ichi; Nakano, Yoshiro; Owada, M. Koji; Kakunaga, Takeo

1986-01-01

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of γ-rays or ultraviolet irradiation was as high as those of their parental fibroblasts. (Auth.)

Genetic influences on ovulation of primary oocytes in LT/Sv strain mice.

Science.gov (United States)

Everett, Clare A; Auchincloss, Catherine A; Kaufman, Matthew H; Abbott, Catherine M; West, John D

2004-11-01

A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.

Recombinant allergy vaccines based on allergen-derived B cell epitopes.

Science.gov (United States)

Valenta, Rudolf; Campana, Raffaela; Niederberger, Verena

2017-09-01

Immunoglobulin E (IgE)-associated allergy is the most common immunologically-mediated hypersensitivity disease. It affects more than 25% of the population. In IgE-sensitized subjects, allergen encounter can causes a variety of symptoms ranging from hayfever (allergic rhinoconjunctivitis) to asthma, skin inflammation, food allergy and severe life-threatening anaphylactic shock. Allergen-specific immunotherapy (AIT) is based on vaccination with the disease-causing allergens. AIT is an extremely effective, causative and disease-modifying treatment. However, administration of natural allergens can cause severe side effects and the quality of natural allergen extracts limits its application. Research in the field of molecular allergen characterization has allowed deciphering the molecular structures of the disease-causing allergens and it has become possible to engineer novel molecular allergy vaccines which precisely target the mechanisms of the allergic immune response and even appear suitable for prophylactic allergy vaccination. Here we discuss recombinant allergy vaccines which are based on allergen-derived B cell epitopes regarding their molecular and immunological properties and review the results obtained in clinical studies with this new type of allergy vaccines. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome.

Science.gov (United States)

Cottingham, Matthew G; Gilbert, Sarah C

2010-09-01

The non-replicating poxviral vector modified vaccinia virus Ankara (MVA) is currently a leading candidate for development of novel recombinant vaccines against globally important diseases. The 1980s technology for making recombinant MVA (and other poxviruses) is powerful and robust, but relies on rare recombination events in poxviral-infected cells. In the 21st century, it has become possible to apply bacterial artificial chromosome (BAC) technology to poxviruses, as first demonstrated by B. Moss' lab in 2002 for vaccinia virus. A similar BAC clone of MVA was subsequently derived, but while recombination-mediated genetic engineering for rapid production was used of deletion mutants, an alternative method was required for efficient insertion of transgenes. Furthermore "markerless" viruses, which carry no trace of the selectable marker used for their isolation, are increasingly required for clinical trials, and the viruses derived via the new method contained the BAC sequence in their genomic DNA. Two methods are adapted to MVA-BAC to provide more rapid generation of markerless recombinants in weeks rather than months. "En passant" recombineering is applied to the insertion of a transgene expression cassette and the removal of the selectable marker in bacteria; and a self-excising variant of MVA-BAC is constructed, in which the BAC cassette region is rapidly and efficiently lost from the viral genome following rescue of the BAC into infectious virus. These methods greatly facilitate and accelerate production of recombinant MVA, including markerless constructs. Copyright 2010 Elsevier B.V. All rights reserved.

Assessment of 40K levels in foodstuffs by gamma spectrometry

International Nuclear Information System (INIS)

Chinnaesakki, S.; Sartandel, S.J.; Bara, S.V.; Krishna, N.S.; Vinod Kumar, A.; Tripathi, R.M.; Puranik, V.D.

2012-01-01

Potassium-40 ( 40 K) is a naturally occurring radioactive isotope of potassium. Radioactive potassium-40 comprises a very small fraction(0.012%) of naturally occurring potassium, which is an element found in large amounts throughout nature. The half-life of 40 K is 1.3 billion years, and it decays to calcium-40 ( 40 Ca) by emitting a beta particle (89 %) and to the argon-40 ( 40 Ar) gas by electron capture with the emission of an energetic gamma ray of energy 1460 keV (10.7%). Humans require potassium to sustain biological processes, with most including 40 K being almost completely absorbed upon ingestion, moving quickly from the gastrointestinal tract to the blood stream (ANL-EVS, 2007). The body content of potassium is under homeostatic control. The total annual effective dose due to inhalation and ingestion of terrestrial radionuclides is assessed to be 0.29 mSv, of which 0.17mSv is due to 40 K and 0.12 mSv to the long-lived radionuclides in the uranium and thorium series (UNSCEAR 2008). Hence, measurement of 40 K in foodstuff is very much essential. This paper discusses the measurement of 40 K in foodstuffs samples collected in and around the new Bhabha Atomic Research Centre campus, Vishakhapatnam, Andhra Pradesh. This study was carried out as part of the baseline radiological monitoring

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Directory of Open Access Journals (Sweden)

Eva Weber

Full Text Available Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3 (- as the first ionic interaction partner, but not necessarily for Ca(2+. The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

Science.gov (United States)

Arthur, Kelly K; Kendrick, Brent S; Gabrielson, John P

2015-01-01

The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market. © 2015 Elsevier Inc. All rights reserved.

MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

Science.gov (United States)

Gradia, Scott D; Ishida, Justin P; Tsai, Miaw-Sheue; Jeans, Chris; Tainer, John A; Fuss, Jill O

2017-01-01

Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done. © 2017 Elsevier Inc. All rights reserved.

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase

Energy Technology Data Exchange (ETDEWEB)

Zemla, A; Lang, D; Kostova, T; Andino, R; Zhou, C

2010-11-29

Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory - still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could overcome these difficulties and facilitate the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV, a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus and demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique or that shared structural similarity with structures that are distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected

40 CFR 721.9079 - Dihydro quinacridone derivative (generic).

Science.gov (United States)

2010-07-01

...) Industrial, commercial, and consumer activities. Requirements as specified in § 721.80(s) (1,000 kg). (ii... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Dihydro quinacridone derivative (generic). 721.9079 Section 721.9079 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED...

Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

Science.gov (United States)

Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

2016-11-01

Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Genome-Wide Comparative Functional Analyses Reveal Adaptations of Salmonella sv. Newport to a Plant Colonization Lifestyle

Directory of Open Access Journals (Sweden)

Marcos H. de Moraes

2018-05-01

Full Text Available Outbreaks of salmonellosis linked to the consumption of vegetables have been disproportionately associated with strains of serovar Newport. We tested the hypothesis that strains of sv. Newport have evolved unique adaptations to persistence in plants that are not shared by strains of other Salmonella serovars. We used a genome-wide mutant screen to compare growth in tomato fruit of a sv. Newport strain from an outbreak traced to tomatoes, and a sv. Typhimurium strain from animals. Most genes in the sv. Newport strain that were selected during persistence in tomatoes were shared with, and similarly selected in, the sv. Typhimurium strain. Many of their functions are linked to central metabolism, including amino acid biosynthetic pathways, iron acquisition, and maintenance of cell structure. One exception was a greater need for the core genes involved in purine metabolism in sv. Typhimurium than in sv. Newport. We discovered a gene, papA, that was unique to sv. Newport and contributed to the strain’s fitness in tomatoes. The papA gene was present in about 25% of sv. Newport Group III genomes and generally absent from other Salmonella genomes. Homologs of papA were detected in the genomes of Pantoea, Dickeya, and Pectobacterium, members of the Enterobacteriacea family that can colonize both plants and animals.

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

NARCIS (Netherlands)

Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

SequenceLDhot: detecting recombination hotspots.

Science.gov (United States)

Fearnhead, Paul

2006-12-15

There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (maths.lancs.ac.uk/~fearnhea/Hotspot.

Toxicological Evaluation of Lactase Derived from Recombinant Pichia pastoris

Science.gov (United States)

Liu, Yifei; Chen, Delong; Luo, Yunbo; Huang, Kunlun; Zhang, Wei; Xu, Wentao

2014-01-01

A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system. PMID:25184300

Toxicological evaluation of lactase derived from recombinant Pichia pastoris.

Directory of Open Access Journals (Sweden)

Shiying Zou

Full Text Available A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50 based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.

Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Science.gov (United States)

Dunn, Amy R; Stout, Kristen A; Ozawa, Minagi; Lohr, Kelly M; Hoffman, Carlie A; Bernstein, Alison I; Li, Yingjie; Wang, Minzheng; Sgobio, Carmelo; Sastry, Namratha; Cai, Huaibin; Caudle, W Michael; Miller, Gary W

2017-03-14

Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect

NARCIS (Netherlands)

Mathijssen, N.C.J.; Masereeuw, R.; Holme, P.A.; Kraaij, M.G.J. van; Laros, B.A.P.; Peyvandi, F.; Heerde, W.L. van

2013-01-01

INTRODUCTION: Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. MATERIALS AND METHODS: Ten factor VII deficient patients

Visualization of SV2A conformations in situ by the use of Protein Tomography

International Nuclear Information System (INIS)

Lynch, Berkley A.; Matagne, Alain; Braennstroem, Annika; Euler, Anne von; Jansson, Magnus; Hauzenberger, Elenor; Soederhaell, J. Arvid

2008-01-01

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography TM . We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins

Aanvullend commentaar op art. 511a Sv

NARCIS (Netherlands)

van Laanen, F.; Melai, A.L.; Groenhuijsen, M.S.

2005-01-01

Schr. geeft een actueel wetenschappelijk commentaar op art. 511a Sv. Daarin komt met name de hedendaagse werkingssfeer aan de orde van deze bepaling, waarin is gesteld dat de berechting van strafbare feiten die ingevolge enige wet aan de burgerlijke rechter is opgedragen, ter terechtzitting van de

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

Science.gov (United States)

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

Human histologic evaluation of anorganic bovine bone mineral combined with recombinant human platelet-derived growth factor BB in maxillary sinus augmentation: case series study.

Science.gov (United States)

Nevins, Myron; Garber, David; Hanratty, James J; McAllister, Bradley S; Nevins, Marc L; Salama, Maurice; Schupbach, Peter; Wallace, Steven; Bernstein, Simon M; Kim, David M

2009-12-01

The objective of this proof-of-principle study was to examine the potential for improved bone regenerative outcomes in maxillary sinus augmentation procedures when recombinant human platelet-derived growth factor BB (0.3 mg/mL) is combined with particulate anorganic bovine bone mineral. The surgical outcomes in all treated sites were uneventful at 6 to 8 months, with sufficient regenerated bone present to allow successful placement of maxillary posterior implants. Large areas of dense, well-formed lamellar bone were seen throughout the intact core specimens in more than half of the grafted sites. Abundant numbers of osteoblasts were noted in concert with significant osteoid in all sites, indicating ongoing osteogenesis. A number of cores demonstrated efficient replacement of the normally slowly resorbing anorganic bovine bone mineral matrix particles with newly formed bone when the matrix was saturated with recombinant human platelet-derived growth factor BB.

Radiation Doses to Hanford Workers from Natural Potassium-40

Energy Technology Data Exchange (ETDEWEB)

Strom, Daniel J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lynch, Timothy P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Weier, Dennis R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2009-02-01

The chemical element potassium is an essential mineral in people and is subject to homeostatic regulation. Natural potassium comprises three isotopes, ³⁹K, ⁴⁰K, and ⁴¹K. Potassium-40 is radioactive, with a half life of 1.248 billion years. In most transitions, it emits a β particle with a maximum energy of 0.560 MeV, and sometimes a gamma photon of 1.461 MeV. Because it is ubiquitous, ⁴⁰K produces radiation dose to all human beings. This report contains the results of new measurements of ⁴⁰K in 248 adult females and 2,037 adult males performed at the Department of Energy Hanford Site in 2006 and 2007. Potassium concentrations diminish with age, are generally lower in women than in men, and decrease with body mass index (BMI). The average annual effective dose from ⁴⁰K in the body is 0.149 mSv y^-1 for men and 0.123 mSv y^-1 women respectively. Averaged over both men and women, the average effective dose per year is 0.136 mSv y^-1. Calculated effective doses range from 0.069 to 0.243 mSv y^-1 for adult males, and 0.067 to 0.203 mSv y^-1 for adult females, a roughly three-fold variation for each gender. The need for dosimetric phantoms with a greater variety of BMI values should be investigated. From our data, it cannot be determined whether the potassium concentration in muscle in people with large BMI values differs from that in people with small BMI values. Similarly, it would be important to know the potassium concentration in other soft tissues, since much of the radiation dose is due to beta radiation, in which the source and target tissues are the same. These uncertainties should be evaluated to determine their consequences for dosimetry.

Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

Science.gov (United States)

Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

2012-11-01

Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

Derivation of residual radionuclide inventory guidelines for implace closure of high-level waste tanks

International Nuclear Information System (INIS)

Yuan, L.; Yuan, Y.

1999-01-01

Residual radionuclide inventory guidelines were derived for the high-level waste tanks at a vitrification facility. The decommissioning scenario assumed for this derivation was that the tanks were to be stabilized at the present locations and the site is released for unrestricted use following a 100-year institutional control period. It was assumed that loss of institutional control would occur at 100-years following tank closure. The derivation of the residual radionuclide inventory guidelines was based on the requirement that the effective dose equivalent (EDE) to a hypothetical individual who lives in the vicinity of the site should not exceed a dose of 0.15 mSv/yr off-site and 5 mSv/yr on-site following closure of the tanks. The RESRAD computer code, modified for exposure scenarios specific for the site, was used for this evaluation. The results of the derivation indicate that the allowable off-site dose limit will not be exceeded. The estimated potential doses to individuals using water offsite from a creek are negligibly small fractions of the 0.15 mSv/yr allowable dose limit. With an assumed 3% heel remaining in the tanks, the estimated peak dose rate for the future offsite water user is about 0.00025 mSv/yr. The residual radionuclide inventory guidelines derived based on potential doses to the on-site resident farmer indicate that, with the exception of Tc-99 and C-14, a 3% heel remaining in the tanks would not result in doses exceeding the 5 mSv/yr allowable dose limit. For this on-site exposure scenario, the peak dose rates occur at about 2000 years after tank closure. The peak dose rate is calculated to be 25 mSv/yr, with greater than 99% produced by four radionuclides: C-14, Tc-99, Np-237, and Am-241. Ingestion of contaminated vegetation contributes most (90%) of the peak dose. Since the inventories used for the derivation are mostly estimated from fuel depletion calculations. There is a need to determine further the actual inventories of these

Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

Science.gov (United States)

Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

1995-02-01

The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

Science.gov (United States)

Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

2015-09-18

BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

Directory of Open Access Journals (Sweden)

Emily Qualls-Creekmore

2017-07-01

Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

Production, purification and characterization of two recombinant ...

African Journals Online (AJOL)

STORAGESEVER

2008-06-17

Jun 17, 2008 ... Two recombinant DNA-derived variants of ovine growth hormone were produced, purified, characterized and compared with the authentic pituitary derived GH. The variants oGH3 and oGH5 were isolated by differential centrifugation method and were purified after refolding by ion-exchange.

Activity of recombinant factor VIIa under different conditions in vitro

DEFF Research Database (Denmark)

Bladbjerg, Else-Marie; Jespersen, Jørgen

2008-01-01

Recombinant activated factor VII (NovoSeven; Novo Nordisk A/S, Måløv, Denmark) is an effective drug for treatment of bleeding in patients with haemophilia A or B and inhibitors. Little is known about physiological conditions influencing the efficacy of recombinant activated factor VII. We...... investigated the in-vitro effects of pH, temperature, and haemodilution on the activity of recombinant activated factor VII. Samples from eight healthy volunteers were spiked with recombinant activated factor VII (final concentration 1.7 microg/ml) and adjusted to pH 6.0, 6.5, 7.0, and 7.4 or analysed at 30......, 33, 37, and 40 degrees C, or diluted 0, 10, 20, 40, and 60% with dextran before analysis. Samples were analysed as rotational thromboelastometry in whole blood (clotting time, clot formation time, and maximum clot firmness) with and without Innovin (tissue factor), and as factor VII coagulant...

New rifamycins produced by a recombinant strain of Nocardia mediterranei.

Science.gov (United States)

Schupp, T; Traxler, P; Auden, J A

1981-08-01

A recombinant strain of Nocardia mediterranei was found to produce a number of new rifamycins which are structurally related to rifamycin S, rifamycin W and rifamycin G. This strain was derived from two Nocardia mediterranei mutants by intraspecific recombination.

SV-BR-1-GM, a Clinically Effective GM-CSF-Secreting Breast Cancer Cell Line, Expresses an Immune Signature and Directly Activates CD4+ T Lymphocytes

Directory of Open Access Journals (Sweden)

Markus D. Lacher

2018-05-01

Full Text Available Targeted cancer immunotherapy with irradiated, granulocyte–macrophage colony-stimulating factor (GM-CSF-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862, a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB, in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B, ADA (encoding adenosine deaminase, ADGRE5 (CD97, CD58 (LFA3, CD74 (encoding invariant chain and CLIP, CD83, CXCL8 (IL8, CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele treated with

Photoionization and electron-ion recombination of Cr I

International Nuclear Information System (INIS)

Nahar, Sultana N.

2009-01-01

Using the unified method, the inverse processes of photoionization and electron-ion recombination are studied in detail for neutral chromium, (CrI+hν↔CrII+e), for the ground and excited states. The unified method based on close-coupling approximation and R-matrix method (i) subsumes both the radiative recombination (RR) and dielectronic recombination (DR) for the total rate and (ii) provides self-consistent sets of photoionization cross sections σ PI and recombination rates α RC . The present results show in total photoionization of the ground and excited states an enhancement in the background at the first excited threshold, 3d 4 4s 5 D state of the core. One prominent phot-excitation-of-core (PEC) resonance due to one dipole allowed transition ( 6 S- 6 P o ) in the core is found in the photoionization cross sections of most of the valence electron excited states. Structures in the total and partial photoionization, for ionization into various excited core states and ground state only, respectively, are demonstrated. Results are presented for the septet and quintet states with n≤10 and l≤9 of Cr I. These states couple to the core ground state 6 S and contribute to the recombination rates. State-specific recombination rates are also presented for these states and their features are illustrated. The total recombination rate shows two DR peaks, one at a relatively low temperature, at 630 K, and the other around 40,000 K. This can explain existence of neutral Cr in interstellar medium. Calculations were carried out in LS coupling using a close-coupling wave function expansion of 40 core states. The results illustrate the features in the radiative processes of Cr I and provide photoionization cross sections and recombination rates with good approximation for this astrophysically important ion.

Derivation of recommended limits for radionuclide contamination of foods by the FAO

International Nuclear Information System (INIS)

Wirth, E.; Mueller, M.K.

1986-01-01

As a consequence of the reactor accident at Chernobyl, USSR, various countries defined limits for radioactive contamination of foods at different levels. These limits ranged from a few Bq iodine 131 or cesium 134 + 137/kg (Malaysia and Canada) to more than 1000 Bq/kg (Great Britain and France). These variations in limits hindered the movement of foods in international trade. For this reason the FAO convened an Expert Consultation to derive 'action levels' below which neither intervention nor constraint would be justified in terms of international movement and trade in food and drink. These limits are to find application in cases of widespread environmental contamination after an accidental release of radionuclides. They are not to be applied in cases of local release, e.g. in the vicinity of nuclear facilities. The derivation of 'action levels' by the FAO was based on the recommendations issued by the International Commission of Radiological Protection (ICRP) in May 1985. According to this recommendation the committed dose equivalent for the whole body should not exceed 5 mSv in the first and 1 mSv in the consecutive years. For radionuclides that preferentially irradiate individual organs, e.g. I131 in the thyroid, the dose equivalent to a specified organ may be used to derive limiting values. For individual organs limiting doses of 50 mSv/a and 10 mSv/a respectively were chosen

Derivation of recommended limits for radionuclide contamination of foods by the FAO

Energy Technology Data Exchange (ETDEWEB)

Wirth, E; Mueller, M K [Institute for Radiation Hygiene, Federal Health Office, Neuherberg (Germany)

1986-07-01

As a consequence of the reactor accident at Chernobyl, USSR, various countries defined limits for radioactive contamination of foods at different levels. These limits ranged from a few Bq iodine 131 or cesium 134 + 137/kg (Malaysia and Canada) to more than 1000 Bq/kg (Great Britain and France). These variations in limits hindered the movement of foods in international trade. For this reason the FAO convened an Expert Consultation to derive 'action levels' below which neither intervention nor constraint would be justified in terms of international movement and trade in food and drink. These limits are to find application in cases of widespread environmental contamination after an accidental release of radionuclides. They are not to be applied in cases of local release, e.g. in the vicinity of nuclear facilities. The derivation of 'action levels' by the FAO was based on the recommendations issued by the International Commission of Radiological Protection (ICRP) in May 1985. According to this recommendation the committed dose equivalent for the whole body should not exceed 5 mSv in the first and 1 mSv in the consecutive years. For radionuclides that preferentially irradiate individual organs, e.g. I131 in the thyroid, the dose equivalent to a specified organ may be used to derive limiting values. For individual organs limiting doses of 50 mSv/a and 10 mSv/a respectively were chosen.

The extent and importance of intragenic recombination

Directory of Open Access Journals (Sweden)

de Silva Eric

2004-11-01

Full Text Available Abstract We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

Directory of Open Access Journals (Sweden)

Marcela Cristina Correa de Freitas

2014-06-01

Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

Antička baština samostana sv. Frane u Splitu

OpenAIRE

Cambi, Nenad

2005-01-01

Na mjestu današnjega franjevačkog samostana sv. Frane u Splitu otkriveno je nekoliko starokršćanskih spomenika: dva natpisa (danas izgubljena), sarkofag s prikazom motiva Prelaska Izraelaca preko Crvenoga mora, fragment s prikazom Krista i apostola te najvjerojatnije i motivom Davida i Golijata, pilastar s urezanim križem, fragment akroterija sarkofaga s križem te omanji fragment pluteja s ljiljanom. Današnja crkva sv. Frane uništila je prethodni samostan i crkvu, koje je u XI. st. podigao sp...

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

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Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

2002-01-01

Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

Characterization of gastric adenocarcinoma cell lines established from CEA424/SV40 T antigen-transgenic mice with or without a human CEA transgene

International Nuclear Information System (INIS)

Nöckel, Jessica; Engel, Natasja K van den; Winter, Hauke; Hatz, Rudolf A; Zimmermann, Wolfgang; Kammerer, Robert

2006-01-01

Gastric carcinoma is one of the most frequent cancers worldwide. Patients with gastric cancer at an advanced disease stage have a poor prognosis, due to the limited efficacy of available therapies. Therefore, the development of new therapies, like immunotherapy for the treatment of gastric cancer is of utmost importance. Since the usability of existing preclinical models for the evaluation of immunotherapies for gastric adenocarcinomas is limited, the goal of the present study was to establish murine in vivo models which allow the stepwise improvement of immunotherapies for gastric cancer. Since no murine gastric adenocarcinoma cell lines are available we established four cell lines (424GC, mGC3, mGC5, mGC8) from spontaneously developing tumors of CEA424/SV40 T antigen (CEA424/Tag) mice and three cell lines derived from double-transgenic offsprings of CEA424/Tag mice mated with human carcinoembryonic antigen (CEA)-transgenic (CEA424/Tag-CEA) mice (mGC2 CEA , mGC4 CEA , mGC11 CEA ). CEA424/Tag is a transgenic C57BL/6 mouse strain harboring the Tag under the control of a -424/-8 bp CEA gene promoter which leads to the development of invasive adenocarcinoma in the glandular stomach. Tumor cell lines established from CEA424/Tag-CEA mice express the well defined tumor antigen CEA under the control of its natural regulatory elements. The epithelial origin of the tumor cells was proven by morphological criteria including the presence of mucin within the cells and the expression of the cell adhesion molecules EpCAM and CEACAM1. All cell lines consistently express the transgenes CEA and/or Tag and MHC class I molecules leading to their susceptibility to lysis by Tag-specific CTL in vitro. Despite the presentation of CTL-epitopes derived from the transgene products the tumor cell lines were tumorigenic when grafted into C57BL/6, CEA424/Tag or CEA424/Tag-CEA-transgenic hosts and no significant differences in tumor take and tumor growth were observed in the different hosts

The Recombination Landscape in Wild House Mice Inferred Using Population Genomic Data.

Science.gov (United States)

Booker, Tom R; Ness, Rob W; Keightley, Peter D

2017-09-01

Characterizing variation in the rate of recombination across the genome is important for understanding several evolutionary processes. Previous analysis of the recombination landscape in laboratory mice has revealed that the different subspecies have different suites of recombination hotspots. It is unknown, however, whether hotspots identified in laboratory strains reflect the hotspot diversity of natural populations or whether broad-scale variation in the rate of recombination is conserved between subspecies. In this study, we constructed fine-scale recombination rate maps for a natural population of the Eastern house mouse, Mus musculus castaneus We performed simulations to assess the accuracy of recombination rate inference in the presence of phase errors, and we used a novel approach to quantify phase error. The spatial distribution of recombination events is strongly positively correlated between our castaneus map, and a map constructed using inbred lines derived predominantly from M. m. domesticus Recombination hotspots in wild castaneus show little overlap, however, with the locations of double-strand breaks in wild-derived house mouse strains. Finally, we also find that genetic diversity in M. m. castaneus is positively correlated with the rate of recombination, consistent with pervasive natural selection operating in the genome. Our study suggests that recombination rate variation is conserved at broad scales between house mouse subspecies, but it is not strongly conserved at fine scales. Copyright © 2017 by the Genetics Society of America.

Utilization of plant-derived recombinant human β-defensins (hBD-1 and hBD-2) for averting salmonellosis.

Science.gov (United States)

Patro, Sunita; Maiti, Soumitra; Panda, Santosh Kumar; Dey, Nrisingha

2015-04-01

We describe the use of plant-made β-defensins as effective antimicrobial substances for controlling salmonellosis, a deadly infection caused by Salmonella typhimurium (referred to further as S. typhi). Human β-defensin-1 (hBD-1) and -2 (hBD-2) were expressed under the control of strong constitutive promoters in tobacco plants, and bio-active β-defensins were successfully extracted. In the in vitro studies, enriched recombinant plant-derived human β-defensin-1 (phBD-1) and -2 (phBD-2) obtained from both T1 and T2 transgenic plants showed significant antimicrobial activity against Escherichia coli and S. typhi when used individually and in various combinations. The 2:1 peptide combination of phBD-1:phBD-2 with peptides isolated from T1-and T2-generation plants reduced the growth of S. typhi by 96 and 85 %, respectively. In vivo studies employing the mouse model (Balb/c) of Salmonella infection clearly demonstrated that the administration of plant-derived defensins individually and in different combinations enhanced the mean survival time of Salmonella-infected animals. When treatment consisted of the 2:1 phBD-1:phBD-2 combination, approximately 50 % of the infected mice were still alive at 206 h post-inoculation; the lowest number of viable S. typhi was observed in the liver and spleen of infected animals. We conclude that plant-made recombinant β-defensins (phBD-1 and phBD-2) are promising antimicrobial substances and have the potential to become additional tools against salmonellosis, particularly when used in combination.

The archetype enhancer of simian virus 40 DNA is duplicated during virus growth in human cells and rhesus monkey kidney cells but not in green monkey kidney cells

International Nuclear Information System (INIS)

O'Neill, Frank J.; Greenlee, John E.; Carney, Helen

2003-01-01

Archetype SV40, obtained directly from its natural host, is characterized by a single 72-bp enhancer element. In contrast, SV40 grown in cell culture almost invariably exhibits partial or complete duplication of the enhancer region. This distinction has been considered important in studies of human tumor material, since SV40-associated tumor isolates have been described having a single enhancer region, suggesting natural infection as opposed to possible contamination by laboratory strains of virus. However, the behavior of archetypal SV40 in cultured cells has never been methodically studied. In this study we reengineered nonarchetypal 776-SV40 to contain a single 72-bp enhancer region and used this reengineered archetypal DNA to transfect a number of simian and human cell lines. SV40 DNA recovered from these cells was analyzed by restriction endonuclease analysis, PCR, and DNA sequencing. Reengineered archetype SV40 propagated in green monkey TC-7 or BSC-1 kidney cells remained without enhancer region duplication even after extensive serial virus passage. Archetype SV40 grown in all but one of the rhesus or human cell lines initially appeared exclusively archetypal. However, when virus from these cell types was transferred to green monkey cells, variants with partial enhancer duplication appeared after as little as a single passage. These findings suggest (1) that virus with a single 72-bp enhancer may persist in cultured cells of simian and human origin; (2) that variants with partially duplicated enhancer regions may arise within cell lines in quantities below limits of detection; (3) that these variants may enjoy a selective advantage in cell types other than those from which they arose (e.g., green monkey kidney cells); and (4) that certain cell lines may support a selective growth advantage for the variants without supporting their formation. Our data indicate that enhancer duplication may also occur in human as well as rhesus kidney cells. Thus, detection of

Vaccine platform recombinant measles virus.

Science.gov (United States)

Mühlebach, Michael D

2017-10-01

The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

Studying of the standardization principles of pharmacological activity of recombinant erythropoietin preparations

OpenAIRE

A. K. Yakovlev; L. A. Gayderova; N. A. Alpatova; T. N. Lobanova; E. L. Postnova; E. I. Yurchikova; T. A. Batuashvili; R. A. Volkova; V. N. Podkuiko; Yu. V. Olefir

2016-01-01

Analysis of the publications devoted to the structure, functions, mechanism of action of erythropoietin is given in the article. Erythropoietin preparations derived from recombinant DNA technology are a mixture of isoforms with different biological activity, which determine the biological properties pharmacological activity, pharmacokinetics, efficacy and safety of medicinal product. Erythropoietin preparations derived by using recombinant DNA technology are a mixture of isoforms with differe...

Validity and reliability of the Brazilian version of Yale-Brown obsessive compulsive scale-shopping version (YBOCS-SV).

Science.gov (United States)

Leite, Priscilla Lourenço; Filomensky, Tatiana Zambrano; Black, Donald W; Silva, Adriana Cardoso

2014-08-01

The Yale-Brown Obsessive Compulsive Scale-Shopping Version (YBOCS-SV) is considered the gold standard in the assessment of shopping severity. It is designed to assess cognitions and behaviors relating to compulsive buying behavior. The present study aims to assess the validity of the Brazilian version of this scale. For the study, composed the sample 610 participants: 588 subjects of a general population and 22 compulsive buyers. Factorial analysis was performed to assess the relations and the correlation between the YBOCS-SV, the Compulsive Buying Scale (CBS), and Richmond Compulsive Buying Scale (RCBS), was assessed using Pearson coefficient, for study of convergent and divergent validity. Cronbach's alpha coefficients were used to assess internal consistency. The results show good to excellent psychometric parameters for the YBOCS-SV in its Brazilian version. With regard to correlations, the YBOCS-SV is inversely and proportionally correlated with CBS and the RCBS, indicating that the YBOCS-SV is an excellent instrument for screening compulsive buying. The YBOCS-SV presented high alpha coefficient of Cronbach's alpha (0.92), demonstrating good reliability. The Brazilian version of the YBOCS-SV is indicated to diagnose compulsive buying disorder, and likely use for the purposes intended in the Brazilian population. Copyright © 2014 Elsevier Inc. All rights reserved.

Modelling of the operational behaviour of passive autocatalytic recombiners

International Nuclear Information System (INIS)

Schwarz, Ulrich

2011-01-01

Due to severe accidents in nuclear power plants, a significant amount of hydrogen can be produced. In pressurized water reactors, a possible and wide-spread measurement is the use of auto-catalytic recombiners. There are numerous numerical models describing the operational behaviour of recombiners for containment codes. The numerical model REKO-DIREKT was developed at the Forschungszentrum Juelich. This model describes the chemical reaction on the catalytic sheets by a physical model, as opposed to the usual codes based on empirical correlations. Additionally, there have been experimental studies concerning the catalytic recombination of hydrogen since the 1990s. The aim of this work is the further development of the program REKO-DIREKT to an independent recombiner model for severe accident and containment codes. Therefore, the catalyst model already existed has been submitted by a parameter optimization with an experimental database expanded during this work. In addition, a chimney model has been implemented which allows the calculation of the free convection flow through the recombiner housing due to the exothermal reaction. This model has been tested by experimental data gained by a recently built test facility. The complete recombiner model REKO-DIREKT has been validated by data from literature. Another aim of this work is the derivation of the reaction kinetics for recombiner designs regarding future reactor concepts. Therefore, experimental studies both on single catalytic coated meshes as well as on two meshes installed in a row have been performed in laboratory scale. By means of the measured data, a theoretical approach for the determination of the reaction rate has been derived.

Dynamic VaR Measurement of Gold Market with SV-T-MN Model

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Fenglan Li

2017-01-01

Full Text Available VaR (Value at Risk in the gold market was measured and predicted by combining stochastic volatility (SV model with extreme value theory. Firstly, for the fat tail and volatility persistence characteristics in gold market return series, the gold price return volatility was modeled by SV-T-MN (SV-T with Mixture-of-Normal distribution model based on state space. Secondly, future sample volatility prediction was realized by using approximate filtering algorithm. Finally, extreme value theory based on generalized Pareto distribution was applied to measure dynamic risk value (VaR of gold market return. Through the proposed model on the price of gold, empirical analysis was investigated; the results show that presented combined model can measure and predict Value at Risk of the gold market reasonably and effectively and enable investors to further understand the extreme risk of gold market and take coping strategies actively.

Dynamics and Predictive Potential of Antibodies against Insect-Derived Recombinant Leishmania infantum Proteins during Chemotherapy of Naturally Infected Dogs

Science.gov (United States)

Todolí, Felicitat; Galindo, Inmaculada; Gómez-Sebastián, Silvia; Pérez-Filgueira, Mariano; Escribano, José M.; Alberola, Jordi; Rodríguez-Cortés, Alhelí

2010-01-01

A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis. PMID:20439957

Delayed recombination and cosmic parameters

International Nuclear Information System (INIS)

Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

2008-01-01

Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n s , and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z * =1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ε α i <0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

Evolutionary models of early-type contact binary SV Centauri

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Y; Saio, H [Tohoku Univ., Sendai (Japan). Faculty of Science; Sugimoto, Daiichiro

1978-12-01

Models of the early-type contact binary system SV Centauri are computed with a binary-star evolution program. The effects of mass exchange, i.e., the effects of mass acceptance as well as mass loss, are properly included. With the initial masses of the component stars as 12.4 and 8.0 M sub(solar mass), the following observed configurations are well reproduced; the component stars are definitely in contact and the rate of mass exchange is 4 x 10/sup -4/ M sub(solar mass)yr/sup -1/. The more massive component is less luminous and has a lower effective temperature. Such features are also reproduced quantitatively. Agreement of the computed models with observation indicates that the binary system SV Cen is actually in the phase of rapid mass exchange preceding the mass-ratio reversal.

Differential protein expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63-family of proteins in replicative senescence.

Science.gov (United States)

Djelloul, Siham; Tarunina, Marina; Barnouin, Karin; Mackay, Alan; Jat, Parmjit S

2002-02-07

P53 activity plays a key role in mammalian cells when they undergo replicative senescence at their Hayflick limit. To determine whether p63 proteins, members of the family of p53-related genes, are also involved in this process, we examined their expression in serially passaged rat embryo fibroblasts. Upon senescence, two truncated DeltaNp63 proteins decreased in abundance whereas two TAp63 isoforms accumulated. 2-D gel analysis showed that the DeltaNp63 proteins underwent post-translational modifications in both proliferating and senescent cells. Direct binding of DeltaNp63 proteins to a p53 consensus motif was greater in proliferating cells than senescent cells. In contrast p63alpha isoforms bound to DNA in a p53 dependent manner and this was higher in senescent cells than proliferating cells. An interaction of p63alpha proteins with SV40 large tumour antigen was also detected and ectopic expression of DeltaNp63alpha can extend the lifespan of rat embryo fibroblasts. Taken together the results indicate that p63 proteins may play a role in replicative senescence either by competition for p53 DNA binding sites or by direct interaction with p53 protein bound to DNA.

PCR-mediated recombination in amplification products derived from polyploid cotton.

Science.gov (United States)

Richard C. Cronn; M. Cedroni; T. Haselkorn; C. Grover; Jonathan F. Wendel

2002-01-01

PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirements of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more...

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

Science.gov (United States)

Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor

Recombination in hepatitis C virus.

Science.gov (United States)

González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

2011-10-01

Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells.

Science.gov (United States)

Garba, Abubakar; Desmarets, Lowiese M B; Acar, Delphine D; Devriendt, Bert; Nauwynck, Hans J

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes.

Stowaways in the history of science: the case of simian virus 40 and clinical research on federal prisoners at the US National Institutes of Health, 1960.

Science.gov (United States)

Stark, Laura; Campbell, Nancy D

2014-12-01

In 1960, J. Anthony Morris, a molecular biologist at the US National Institutes of Health conducted one of the only non-therapeutic clinical studies of the cancer virus SV40. Morris and his research team aimed to determine whether SV40 was a serious harm to human health, since many scientists at the time suspected that SV40 caused cancer in humans based on evidence from in vivo animal studies and experiments with human tissue. Morris found that SV40 had no significant effect but his claim has remained controversial among scientists and policymakers through the present day--both on scientific and ethical grounds. Why did Morris only conduct one clinical study on the cancer-causing potential of SV40 in healthy humans? We use the case to explain how empirical evidence and ethical imperatives are, paradoxically, often dependent on each other and mutually exclusive in clinical research, which leaves answers to scientific and ethical questions unsettled. This paper serves two goals: first, it documents a unique--and uniquely important--study of clinical research on SV40. Second, it introduces the concept of "the stowaway," which is a special type of contaminant that changes the past in the present moment. In the history of science, stowaways are misfortunes that nonetheless afford research that otherwise would have been impossible specifically by creating new pasts. This case (Morris' study) and concept (the stowaway) bring together history of science and philosophy of history for productive dialog. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

International Nuclear Information System (INIS)

Zamansky, G.B.; Kleinman, L.F.; Black, P.H.; Kaplan, J.C.

1980-01-01

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40(SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The UV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical conditions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line. (orig.)

Role of unsaturated derivatives of spermidine as substrates for spermine synthase and in supporting growth of SV-3T3 cells.

OpenAIRE

Pegg, A E; Nagarajan, S; Naficy, S; Ganem, B

1991-01-01

Synthetic unsaturated analogues of the natural polyamine were examined as possible substrates for spermine synthase and as replacements for spermidine in supporting the growth of SV-3T3 cells. It was found that N-(3-aminopropyl)-1,4-diamino-cis-but-2-ene [the cis isomer of the alkene analogue of spermidine] was a good substrate for spermine synthase, but that the trans isomer [N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and the alkene analogue [N-(3-aminopropyl)-1,4-diaminobut-2-yne] were ...

The monetary value of the man-mSv for Korean NPP radiation workers assessed by the radiation aversion factor

International Nuclear Information System (INIS)

Lee, B. I.; Suh, D. H.; Kim, S. I.; Jeong, M. S.; Lim, Y. K.

2008-01-01

The monetary value of the man-mSv for operators of Korean nuclear power plants (NPPs) was calculated using a radiation aversion factor based on a survey of NPP workers. Initially, the life expectancy in the population is 79.4 y, the average age of cancer occurrence is 60 y, the average annual wage for an electric worker is 56 000 $ y -1 and the nominal risk coefficient induced by radiation is 4.2 E-5 mSv were used to evaluate the basic monetary value (α base) resulting in 45.6 $ mSv -1 . To investigate the degree of radiation aversion, the subject of the investigation was selected as the working radiation workers in 10 NPPs in Korea (Kori 1-2, Yeonggwang 1-3, Ulchin 1-3 and Wolseong 1-2). In August 2010, with the cooperation of KHNP and partner companies, a total of 2500 survey questionnaires to 10 NPPs (or 250 surveys to each NPP) were distributed to currently employed radiation workers. From these, 2157 responses were obtained between August and October 2010. The assessed radiation aversion factor and the monetary value of the man-mSv from the calculated radiation aversion factor were 1.26 and ∼50 $ in the 0-1 mSv range, 1.38 and ∼200 $ in the 1-5 mSv range, 1.52 and ∼1000 $ in the 5-10 mSv range, 1.65 and ∼4000 $ in the 10-20 mSv range and 1.74 and ∼8500 $ >20 mSv. (authors)

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

International Nuclear Information System (INIS)

Chuang, C.-K.; Chen, W.-J.

2009-01-01

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.

Evaluation of the Reference Numerical Parameters of the Monthly Method in ISO 13790 Considering S/V Ratio

Directory of Open Access Journals (Sweden)

Hee-Jeong Kwak

2015-01-01

Full Text Available Many studies have investigated the accuracy of the numerical parameters in the application of the quasi steady-state calculation method. The aim of this study is to derive the reference numerical parameters of the ISO 13790 monthly method by reflecting the surface-to-volume (S/V ratio and the characteristics of the structures. The calculation process was established, and the parameters necessary to derive the reference numerical parameters were calculated based on the input data prepared for the established calculation processes. The reference numerical parameters were then derived through regression analyses of the calculated parameters and the time constant. The parameters obtained from an apartment building and the parameters of the international standard were both applied to the Passive House Planning Package (PHPP and EnergyPlus programs, and the results were analyzed in order to evaluate the validity of the results. The analysis revealed that the calculation results based on the parameters derived from this study yielded lower error rates than those based on the default parameters in ISO 13790. However, the differences were shown to be negligible in the case of high heat capacity.

INTERACTION BETWEEN DIFFERENT MOLECULAR FORMS OF IMMUNOGLOBULIN A AND RECOMBINANT DERIVATIVES POLYPEPTIDES OF BAC RECEPTOR PROTEINS FROM GROUP B STREPTOCOCCI

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A. S. Korzhueva

2008-01-01

Full Text Available Abstract. The article concerns interactions between immunoglobulin A and recombinant P6, P7, P8 polypeptides, designed on the basis of externally localized Bac protein of the Group B streptococci, possessing IgA-binding activity.There is a current demand for immunochemical reagents that are strictly specific for IgA, in order to develop antigenic standards for detection of IgA levels in biological fluids, as well as for affinity purification of IgA and its fragments.To analyze an opportunity of the abovementioned application ways for these proteins, a special study was performed to assay an interaction capability of recombinant P6, P7, P8 polypeptides binding to Fc regions of different IgA forms (serum IgA, secretory IgA, subclasses of serum IgA – IgA1, IgA2. Selectivity of ligand binding was specially confirmed.It was found out that, among three presented polypeptides, the structure of recombinant P6 derivative proved to be optimal for IgA-binding ability of Bac protein.Structural features of IgA-binding fragments of Bac protein, i.e., binding site position on the IgA molecule (proximity to epitopes for three monoclonal antibodies, variability of the site structure, as well as resistance of binding site for P6, P7, P8 in IgA molecule against partial disulfide bonds reduction. (Med. Immunol., vol. 10, N 4-5, pp 327-336.

Dissociation of recombinant prion autocatalysis from infectivity.

Science.gov (United States)

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Measuring marine iron(III) complexes by CLE-AdSV

NARCIS (Netherlands)

Town, R.M.; Leeuwen, van H.P.

2005-01-01

Iron(iii) speciation data, as determined by competitive ligand exchange?adsorptive stripping voltammetry (CLE-AdSV), is reconsidered in the light of the kinetic features of the measurement. The very large stability constants reported for iron(iii) in marine ecosystems are shown to be possibly due to

Somatic mutation and recombination induced with reactor thermal neutrons in Drosophila melanogaster; Mutacion y recombinacion somaticas inducidas con neutrones termicos de reactor en Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Zambrano A, F.; Guzman R, J.; Paredes G, L.; Delfin L, A. [Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

1997-07-01

The SMART test of Drosophila melanogaster was used to quantify the effect over the somatic mutation and recombination induced by thermal and fast neutrons at the TRIGA Mark III reactor of the ININ at the power of 300 k W for times of 30, 60 and 120 minutes with total equivalent doses respectively of 20.8, 41.6 and 83.2 Sv. A linear relation between the radiation equivalent dose and the frequency of the genetic effects such as mutation and recombination was observed. The obtained results allow to conclude that SMART is a sensitive system to the induced damage by neutrons, so this can be used for studying its biological effects. (Author)

Recombination chamber and a measuring system with sensitivity sufficient for in-flight and low-level dosimetry

International Nuclear Information System (INIS)

Zielczynski, M.; Golnik, N.; Shvidkij, S.V.

1996-01-01

A recombination chamber is proposed to be for determination of ambient dose equivalent, H*(10) during high-altitude flights and space missions. Polarizing electrodes of the chamber are supplied from two capacitors permanently connected to the electrodes. Ions, collected on the measuring electrode, charge a measuring capacitor that is also permanently connected to the electrode. The collected charge is proportional to H*(10), so the chamber with only three capacitors creates a whole measuring system. The special procedure was introduced for charging the supplying capacitors and for reading the voltage on the measuring capacitor. The procedure has to be performed in laboratory conditions before and after the flight. The measuring system with recombination chamber of REM-2 type allows to measure the H*(10) above 10 μSv with uncertainly ca. 25% in any field of penetration radiation, with an integration time up to some days. The system has been checked in field conditions. (author). 1 fig

Comprehension of derivational morphemes in words and pseudo-words in semantic variant primary progressive aphasia

Directory of Open Access Journals (Sweden)

Noémie Auclair-Ouellet

2014-04-01

The results of the word condition alone cannot rule out the possibility that errors in the svPPA group were caused by difficulty in understanding words rather than in processing derivational morphemes. However, the lexical context provided in this condition did not speed-up the performance of svPPA individuals as it did in the control group. Most importantly, results from the pseudo-word condition showed that in the svPPA group, the association between the morpheme and its meaning was not performed as readily and reliably as in the control group. These results support the involvement of semantic memory in morphological processing.

Genetics of Genome-Wide Recombination Rate Evolution in Mice from an Isolated Island.

Science.gov (United States)

Wang, Richard J; Payseur, Bret A

2017-08-01

Recombination rate is a heritable quantitative trait that evolves despite the fundamentally conserved role that recombination plays in meiosis. Differences in recombination rate can alter the landscape of the genome and the genetic diversity of populations. Yet our understanding of the genetic basis of recombination rate evolution in nature remains limited. We used wild house mice ( Mus musculus domesticus ) from Gough Island (GI), which diverged recently from their mainland counterparts, to characterize the genetics of recombination rate evolution. We quantified genome-wide autosomal recombination rates by immunofluorescence cytology in spermatocytes from 240 F 2 males generated from intercrosses between GI-derived mice and the wild-derived inbred strain WSB/EiJ. We identified four quantitative trait loci (QTL) responsible for inter-F 2 variation in this trait, the strongest of which had effects that opposed the direction of the parental trait differences. Candidate genes and mutations for these QTL were identified by overlapping the detected intervals with whole-genome sequencing data and publicly available transcriptomic profiles from spermatocytes. Combined with existing studies, our findings suggest that genome-wide recombination rate divergence is not directional and its evolution within and between subspecies proceeds from distinct genetic loci. Copyright © 2017 by the Genetics Society of America.

GABA(A)-benzodiazepine receptor complex sensitivity in 5-HT(1A) receptor knockout mice on a 129/Sv background.

NARCIS (Netherlands)

Pattij, T.; Groenink, L.; Oosting, R.S.; Gugten, J. van der; Maes, R.A.A.; Olivier, B.

2002-01-01

Previous studies in 5-HT(1A) receptor knockout (1AKO) mice on a mixed Swiss Websterx129/Sv (SWx129/Sv) and a pure 129/Sv genetic background suggest a differential gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor complex sensitivity in both strains, independent from the anxious phenotype. To

Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

Directory of Open Access Journals (Sweden)

Kai Fu

Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

The role of recombination in the emergence of a complex and dynamic HIV epidemic

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Morgenstern Burkhard

2010-03-01

Full Text Available Abstract Background Inter-subtype recombinants dominate the HIV epidemics in three geographical regions. To better understand the role of HIV recombinants in shaping the current HIV epidemic, we here present the results of a large-scale subtyping analysis of 9435 HIV-1 sequences that involve subtypes A, B, C, G, F and the epidemiologically important recombinants derived from three continents. Results The circulating recombinant form CRF02_AG, common in West Central Africa, appears to result from recombination events that occurred early in the divergence between subtypes A and G, followed by additional recent recombination events that contribute to the breakpoint pattern defining the current recombinant lineage. This finding also corrects a recent claim that G is a recombinant and a descendant of CRF02, which was suggested to be a pure subtype. The BC and BF recombinants in China and South America, respectively, are derived from recent recombination between contemporary parental lineages. Shared breakpoints in South America BF recombinants indicate that the HIV-1 epidemics in Argentina and Brazil are not independent. Therefore, the contemporary HIV-1 epidemic has recombinant lineages of both ancient and more recent origins. Conclusions Taken together, we show that these recombinant lineages, which are highly prevalent in the current HIV epidemic, are a mixture of ancient and recent recombination. The HIV pandemic is moving towards having increasing complexity and higher prevalence of recombinant forms, sometimes existing as "families" of related forms. We find that the classification of some CRF designations need to be revised as a consequence of (1 an estimated > 5% error in the original subtype assignments deposited in the Los Alamos sequence database; (2 an increasing number of CRFs are defined while they do not readily fit into groupings for molecular epidemiology and vaccine design; and (3 a dynamic HIV epidemic context.

Interfacial recombination at /AlGa/As/GaAs heterojunction structures

Science.gov (United States)

Ettenberg, M.; Kressel, H.

1976-01-01

Experiments were conducted to determine the interfacial recombination velocity at Al0.25Ga0.75As/GaAs and Al0.5Ga0.5As/GaAs heterojunctions. The recombination velocity was derived from a study of the injected minority-carrier lifetime as a function of the junction spacing. It is found that for heterojunction spacings in excess of about 1 micron, the interfacial recombination can be characterized by a surface recombination velocity of 4,000 and 8,000 cm/sec for the two types of heterojunctions, respectively. For double-heterojunction spacings below 1 micron, the constancy of the minority-carrier lifetime suggests that the interfacial recombination velocity decreases effectively. This effect is technologically very important since it makes it possible to construct very low-threshold injection lasers. No such effect is observed in single-heterojunction diodes.

Modulating Excitonic Recombination Effects through One-Step Synthesis of Perovskite Nanoparticles for Light-Emitting Diodes.

Science.gov (United States)

Kulkarni, Sneha A; Muduli, Subas; Xing, Guichuan; Yantara, Natalia; Li, Mingjie; Chen, Shi; Sum, Tze Chien; Mathews, Nripan; White, Tim J; Mhaisalkar, Subodh G

2017-10-09

The primary advantages of halide perovskites for light-emitting diodes (LEDs) are solution processability, direct band gap, good charge-carrier diffusion lengths, low trap density, and reasonable carrier mobility. The luminescence in 3 D halide perovskite thin films originates from free electron-hole bimolecular recombination. However, the slow bimolecular recombination rate is a fundamental performance limitation. Perovskite nanoparticles could result in improved performance but processability and cumbersome synthetic procedures remain challenges. Herein, these constraints are overcome by tailoring the 3 D perovskite as a near monodisperse nanoparticle film prepared through a one-step in situ deposition method. Replacing methyl ammonium bromide (CH 3 NH 3 Br, MABr) partially by octyl ammonium bromide [CH 3 (CH 2 ) 7 NH 3 Br, OABr] in defined mole ratios in the perovskite precursor proved crucial for the nanoparticle formation. Films consisting of the in situ formed nanoparticles displayed signatures associated with excitonic recombination, rather than that of bimolecular recombination associated with 3 D perovskites. This transition was accompanied by enhanced photoluminescence quantum yield (PLQY≈20.5 % vs. 3.40 %). Perovskite LEDs fabricated from the nanoparticle films exhibit a one order of magnitude improvement in current efficiency and doubling in luminance efficiency. The material processing systematics derived from this study provides the means to control perovskite morphologies through the selection and mixing of appropriate additives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced replication of damaged SV40 DNA in carcinogen-treated monkey cells

International Nuclear Information System (INIS)

Maga, J.A.; Dixon, K.

1984-01-01

Treatment of mammalian cells with certain chemical or physical carcinogens prior to infection with ultraviolet-irradiated virus results in enhanced survival or reactivation of the damaged virus. To investigate the molecular basis of this enhanced reactivation (ER), Simian virus 40 DNA replication in carcinogen-treated cells was examined. Treatment of monkey kidney cells with N-acetoxy-2-acetylamino-fluorene or UV radiation 24 h prior to infection with ultraviolet-irradiated Simian virus 40 leads to enhancement of viral DNA replication measured at 36 h after infection by [ 3 H]thymidine incorporation or hybridization. The enhancement of DNA replication is observed when cells are treated from 1 to 60 h before infection or 1 to 16 h after infection. The fact that enhancement is observed also when cells are treated after infection rules out the possiblity that enhancement occurs at the level of adsorption or penetration of the virus. Measurements of the time course of viral DNA replication indicate that pretreatment of cells does not alter the time of onset of viral DNA replication. It is concluded that ER of Simain virus 40 occurs at the level of viral DNA replication. (author)

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik

2013-01-01

Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described....... This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable...

A high-quality narrow passband filter for elastic SV waves via aligned parallel separated thin polymethylmethacrylate plates

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Jun Zhang

2017-08-01

Full Text Available We designed a high-quality filter that consists of aligned parallel polymethylmethacrylate (PMMA thin plates with small gaps for elastic SV waves propagate in metals. Both the theoretical model and the full numerical simulation show the transmission spectrum of the elastic SV waves through such a filter has several sharp peaks with flawless transmission within the investigated frequencies. These peaks can be readily tuned by manipulating the geometry parameters of the PMMA plates. Our investigation finds that the same filter performs well for different metals where the elastic SV waves propagated.

Thermal recombination: Beyond the valence quark approximation

Energy Technology Data Exchange (ETDEWEB)

Mueller, B. [Department of Physics, Duke University, Durham, NC 27708 (United States); Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: fries@physics.umn.edu; Bass, S.A. [Department of Physics, Duke University, Durham, NC 27708 (United States); RIKEN BNL Research Center, Brookhaven National Laboratory, Upton, NY 11973 (United States)

2005-07-07

Quark counting rules derived from recombination models agree well with data on hadron production at intermediate transverse momenta in relativistic heavy-ion collisions. They convey a simple picture of hadrons consisting only of valence quarks. We discuss the inclusion of higher Fock states that add sea quarks and gluons to the hadron structure. We show that, when recombination occurs from a thermal medium, hadron spectra remain unaffected by the inclusion of higher Fock states. However, the quark number scaling for elliptic flow is somewhat affected. We discuss the implications for our understanding of data from the Relativistic Heavy Ion Collider.

Derivation of Intervention levels for Protection of the Public in a Radiological Emergency in Korea

International Nuclear Information System (INIS)

Lee, Jong Tai; Lee, Goan Yup; Khang, Byung Oui; Oh, Ki Hoon; Kim, Chang Kyu

2001-01-01

Intervention levels for protection of the public in a radiological emergency are theoretically derived by the cost-benefit approach with the concept of justification and optimization. Intervention levels on the sheltering, evacuation, temporary relocation and permanent resettlement for protection of the public are estimated with the cost to protective countermeasures and the value from dose averted which are the site specific parameters. As a result, it is confirmed that IAEA guidelines for intervention levels are applicable to the radiological emergency in Korea. Optimum ranges of 5 - 10 mSv/2days for sheltering, 25 - 130 mSv/week for evacuation, 15 - 90 mSv/month for temporary relocation and 600 - 3,500 mSv/lifetime for permanent resettlement for intervention levels are also provided. The result can be applied as useful data to update intervention levels under the theoretical background in Korea

Genetic recombination of Herpes simplex virus, the role of the host cell and UV-irradiation of the virus

International Nuclear Information System (INIS)

Dasgupta, U.B.; Summers, W.C.; Yale Univ., New Haven, CT; Yale Univ., New Haven, CT

1980-01-01

Recombination frequencies for two sets of genetic markers of Herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent. (orig.) [de

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

Science.gov (United States)

Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2016-01-01

Botulinum neurotoxin serotype A1 (BoNT/A1) is one of the most dangerous potential bioterrorism agents, and exerts its action by invading motoneurons. It is also a licensed drug widely used for medical and cosmetic applications. Here we report a 2.0 Å resolution crystal structure of BoNT/A1 receptor-binding domain in complex with its neuronal receptor, the glycosylated human SV2C. We find that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an anti-botulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications to achieve highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors. PMID:27294781

Controlled chondrogenesis from adipose-derived stem cells by recombinant transforming growth factor-β3 fusion protein in peptide scaffolds.

Science.gov (United States)

Zheng, Dong; Dan, Yang; Yang, Shu-hua; Liu, Guo-hui; Shao, Zeng-wu; Yang, Cao; Xiao, Bao-jun; Liu, Xiangmei; Wu, Shuilin; Zhang, Tainjin; Chu, Paul K

2015-01-01

Adipose-derived stem cells (ADSCs) are promising for cartilage repair due to their easy accessibility and chondrogenic potential. Although chondrogenesis of transforming growth factor-β (TGF-β) mediated mesenchymal stem cells (MSCs) is well established in vitro, clinical tissue engineering requires effective and controlled delivery of TGF-β in vivo. In this work, a self-assembled peptide scaffold was employed to construct cartilages in vivo through the chondrogenesis from ADSCs controlled by recombinant fusion protein LAP-MMP-mTGF-β3 that was transfected by lentiviral vectors. During this course, the addition of matrix metalloproteinases (MMPs) can trigger the release of mTGF-β3 from the recombinant fusion protein of LAP-MMP-mTGF-β3 in the combined scaffolds, thus stimulating the differentiation of ADSCs into chondrogenesis. The specific expression of cartilage genes was analyzed by real-time polymerase chain reaction and Western blot. The expression of chondrocytic markers was obviously upregulated to a higher level compared to the one by commonly used TGF-β3 alone. After 3 weeks of in vitro culturing, the hybrids with differentiated chondrogenesis were then injected subcutaneously into nude mice and retrieved after 4 weeks of culturing in vivo. Histological analysis also confirmed that the recombinant fusion protein was more effective for the formation of cartilage matrix than the cases either with TGF-β3 alone or without LAP-MMP-mTGF-β3 (P<0.05). This study demonstrates that controlled local delivery of the LAP-MMP-mTGF-β3 constructs can accelerate differentiation of ADSCs into the cartilage in vivo, which indicates the great potential of this hybrid in rapid therapy of osteoarthritis. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The use of detectors based on ionisation recombination in radiation protection

International Nuclear Information System (INIS)

Sullivan, A.H.

1984-01-01

Intitial recombination of ionisation in a gas depends on the ionisation density and hence on the linear energy transfer along the tracks of charged particles. This effect can be used as a basis for instruments that respond to different types of ionising radiation approximately in the way required by the quality factor-linear energy transfer relation recommended by the ICRP for use in radiation protection. Empirical instruments based on ionisation recombination that have been used for radiation protection measurements are reviewed, and relations are derived from recombination theory that show that the response of such detectors can be readily predicted. The usefulness of recombination instruments in radiation protection is discussed and their advantages and limitations assessed. It is shown that their main application will be as reference instruments against which other detectors can be calibrated. As an extension to using recombination detectors as reference instruments, the feasibility of specifying radiation quality in terms of ionisation recombination is investigated. (author)

Application of SV40 T-transformed human corneal epithelial cells to evaluate potential irritant chemicals for in vitro alternative eye toxicity.

Science.gov (United States)

Kim, Cho-Won; Park, Geon-Tae; Bae, Ok-Nam; Noh, Minsoo; Choi, Kyung-Chul

2016-01-01

Assessment of eye irritation potential is important to human safety, and it is necessary for various cosmetics and chemicals that may contact the human eye. Until recently, the Draize test was considered the standard method for estimating eye irritation, despite its disadvantages such as the need to sacrifice many rabbits for subjective scoring. Thus, we investigated the cytotoxicity and inflammatory response to standard eye irritants using SV40 T-transformed human corneal epithelial (SHCE) cells as a step toward development of an animal-free alternative eye irritation test. MTT and NRU assays of cell viability were performed to investigate the optimal experimental conditions for SHCE cell viability when cells were exposed to sodium dodecyl sulfate (SDS) as a standard eye irritant at 6.25×10(-3) to 1×10(-1)%. Additionally, cell viability of SHCE cells was examined in response to six potential eye irritants, benzalkonium chloride, dimethyl sulfoxide, isopropanol, SDS, Triton X-100 and Tween 20 at 5×10(-3) to 1×10(-1)%. Finally, we estimated the secretion level of cytokines in response to stimulation by eye irritants in SHCE cells. SHCE cells showed a good response to potential eye irritants when the cells were exposed to potential irritants for 10min at room temperature (RT), and cytokine production increased in a concentration-dependent manner, indicating that cytotoxicity and cytokine secretion from SHCE cells may be well correlated with the concentrations of irritants. Taken together, these results suggest that SHCE cells could be an excellent alternative in vitro model to replace in vivo animal models for eye irritation tests. Copyright © 2016 Elsevier Inc. All rights reserved.

PSHA in Israel by using the synthetic ground motions from simulated seismicity: the modified SvE procedure

Science.gov (United States)

Meirova, T.; Shapira, A.; Eppelbaum, L.

2018-05-01

In this study, we updated and modified the SvE approach of Shapira and van Eck (Nat Hazards 8:201-215, 1993) which may be applied as an alternative to the conventional probabilistic seismic hazard assessment (PSHA) in Israel and other regions of low and moderate seismicity where measurements of strong ground motions are scarce. The new computational code SvE overcomes difficulties associated with the description of the earthquake source model and regional ground-motion scaling. In the modified SvE procedure, generating suites of regional ground motion is based on the extended two-dimensional source model of Motazedian and Atkinson (Bull Seism Soc Amer 95:995-1010, 2005a) and updated regional ground-motion scaling (Meirova and Hofstteter, Bull Earth Eng 15:3417-3436, 2017). The analytical approach of Mavroeidis and Papageorgiou (Bull Seism Soc Amer 93:1099-1131, 2003) is used to simulate the near-fault acceleration with the near-fault effects. The comparison of hazard estimates obtained by using the conventional method implemented in the National Building Code for Design provisions for earthquake resistance of structures and the modified SvE procedure for rock-site conditions indicates a general agreement with some perceptible differences at the periods of 0.2 and 0.5 s. For the periods above 0.5 s, the SvE estimates are systematically greater and can increase by a factor of 1.6. For the soft-soil sites, the SvE hazard estimates at the period of 0.2 s are greater than those based on the CB2008 ground-motion prediction equation (GMPE) by a factor of 1.3-1.6. We suggest that the hazard estimates for the sites with soft-soil conditions calculated by the modified SvE procedure are more reliable than those which can be found by means of the conventional PSHA. This result agrees with the opinion that the use of a standard GMPE applying the NEHRP soil classification based on the V s, 30 parameter may be inappropriate for PSHA at many sites in Israel.

Report of recombinant norovirus GII.g/GII.12 in Beijing, China.

Science.gov (United States)

Sang, Shaowei; Zhao, Zhongtang; Suo, Jijiang; Xing, Yubin; Jia, Ning; Gao, Yan; Xie, Lijun; Du, Mingmei; Liu, Bowei; Ren, Shiwang; Liu, Yunxi

2014-01-01

Norovirus (NoV) has been recognized as the most important cause of nonbacterial acute gastroenteritis affecting all age group people in the world. Genetic recombination is a common occurance in RNA viruses and many recombinant NoV strains have been described since it was first reported in 1997. However, the knowledge of recombinant NoV in China is extremely limited. A total of 685 stool specimens were tested for NoV infection from the acute gastroenteritis patients who visited one general hospital in Beijing from April 2009 to November 2011. The virus recombination was identified by constructing phylogenetic trees of two genes, further SimPlot and the maximum chi-square analysis. The overall positive rate was 9.6% (66/685). GII.4 New Orleans 2009 and GII.4 2006b variants were the dominant genotype. Four GII.g/GII.12 and one GII.12/GII.3 recombinant strains were confirmed, and all derived from adult outpatients. The predictive recombination point occurred at the open reading frame (ORF)1/ORF2 overlap. The GII.g ORF1/GII.12ORF2 recombinant has been reported in several countries and it was the first report of this recombinant in China.

Measurement Invariance of the Short Version of the Problematic Mobile Phone Use Questionnaire (PMPUQ-SV) across Eight Languages.

Science.gov (United States)

Lopez-Fernandez, Olatz; Kuss, Daria J; Pontes, Halley M; Griffiths, Mark D; Dawes, Christopher; Justice, Lucy V; Männikkö, Niko; Kääriäinen, Maria; Rumpf, Hans-Jürgen; Bischof, Anja; Gässler, Ann-Kathrin; Romo, Lucia; Kern, Laurence; Morvan, Yannick; Rousseau, Amélie; Graziani, Pierluigi; Demetrovics, Zsolt; Király, Orsolya; Schimmenti, Adriano; Passanisi, Alessia; Lelonek-Kuleta, Bernadeta; Chwaszcz, Joanna; Chóliz, Mariano; Zacarés, Juan José; Serra, Emilia; Dufour, Magali; Rochat, Lucien; Zullino, Daniele; Achab, Sophia; Landrø, Nils Inge; Suryani, Eva; Hormes, Julia M; Terashima, Javier Ponce; Billieux, Joël

2018-06-08

The prevalence of mobile phone use across the world has increased greatly over the past two decades. Problematic Mobile Phone Use (PMPU) has been studied in relation to public health and comprises various behaviours, including dangerous, prohibited, and dependent use. These types of problematic mobile phone behaviours are typically assessed with the short version of the Problematic Mobile Phone Use Questionnaire (PMPUQ⁻SV). However, to date, no study has ever examined the degree to which the PMPU scale assesses the same construct across different languages. The aims of the present study were to (i) determine an optimal factor structure for the PMPUQ⁻SV among university populations using eight versions of the scale (i.e., French, German, Hungarian, English, Finnish, Italian, Polish, and Spanish); and (ii) simultaneously examine the measurement invariance (MI) of the PMPUQ⁻SV across all languages. The whole study sample comprised 3038 participants. Descriptive statistics, correlations, and Cronbach's alpha coefficients were extracted from the demographic and PMPUQ-SV items. Individual and multigroup confirmatory factor analyses alongside MI analyses were conducted. Results showed a similar pattern of PMPU across the translated scales. A three-factor model of the PMPUQ-SV fitted the data well and presented with good psychometric properties. Six languages were validated independently, and five were compared via measurement invariance for future cross-cultural comparisons. The present paper contributes to the assessment of problematic mobile phone use because it is the first study to provide a cross-cultural psychometric analysis of the PMPUQ-SV.

Recombinant Programming

OpenAIRE

Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

2004-01-01

This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

Heterogeneous recombination among Hepatitis B virus genotypes.

Science.gov (United States)

Castelhano, Nadine; Araujo, Natalia M; Arenas, Miguel

2017-10-01

The rapid evolution of Hepatitis B virus (HBV) through both evolutionary forces, mutation and recombination, allows this virus to generate a large variety of adapted variants at both intra and inter-host levels. It can, for instance, generate drug resistance or the diverse viral genotypes that currently exist in the HBV epidemics. Concerning the latter, it is known that recombination played a major role in the emergence and genetic diversification of novel genotypes. In this regard, the quantification of viral recombination in each genotype can provide relevant information to devise expectations about the evolutionary trends of the epidemic. Here we measured the amount of this evolutionary force by estimating global and local recombination rates in >4700 HBV complete genome sequences corresponding to nine (A to I) HBV genotypes. Counterintuitively, we found that genotype E presents extremely high levels of recombination, followed by genotypes B and C. On the other hand, genotype G presents the lowest level, where recombination is almost negligible. We discuss these findings in the light of known characteristics of these genotypes. Additionally, we present a phylogenetic network to depict the evolutionary history of the studied HBV genotypes. This network clearly classified all genotypes into specific groups and indicated that diverse pairs of genotypes are derived from a common ancestor (i.e., C-I, D-E and, F-H) although still the origin of this virus presented large uncertainty. Altogether we conclude that the amount of observed recombination is heterogeneous among HBV genotypes and that this heterogeneity can influence on the future expansion of the epidemic. Copyright © 2017 Elsevier B.V. All rights reserved.

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

International Nuclear Information System (INIS)

Atsmon, Jacob; Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari; Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y.; Bartfeld, Daniel; Shulman, Avidor; Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit; Soreq, Hermona; Shaaltiel, Yoseph

2015-01-01

PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2 min before being exposed to 1.33 × LD 50 and 1.5 × LD 50 of toxin and 10 min after exposure to 1.5 × LD 50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t ½ ) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t ½ in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human study exhibited its safety

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

Energy Technology Data Exchange (ETDEWEB)

Atsmon, Jacob [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y. [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Bartfeld, Daniel [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shulman, Avidor, E-mail: avidors@protalix.com [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Soreq, Hermona [Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Shaaltiel, Yoseph [Protalix Biotherapeutics, Science Park, Carmiel (Israel)

2015-09-15

PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2 min before being exposed to 1.33 × LD{sub 50} and 1.5 × LD{sub 50} of toxin and 10 min after exposure to 1.5 × LD{sub 50} survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t{sub ½}) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t{sub ½} in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human study

Recombinant methods for screening human DNA excision repair proficiency

International Nuclear Information System (INIS)

Athas, W.F.

1988-01-01

A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

Recombiner

International Nuclear Information System (INIS)

Kikuchi, Nobuo.

1983-01-01

Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

High-level secretion of native recombinant human calreticulin in yeast

DEFF Research Database (Denmark)

Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

2015-01-01

, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional...... by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration...... recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts...

Measurement Invariance of the Short Version of the Problematic Mobile Phone Use Questionnaire (PMPUQ-SV across Eight Languages

Directory of Open Access Journals (Sweden)

Olatz Lopez-Fernandez

2018-06-01

Full Text Available The prevalence of mobile phone use across the world has increased greatly over the past two decades. Problematic Mobile Phone Use (PMPU has been studied in relation to public health and comprises various behaviours, including dangerous, prohibited, and dependent use. These types of problematic mobile phone behaviours are typically assessed with the short version of the Problematic Mobile Phone Use Questionnaire (PMPUQ–SV. However, to date, no study has ever examined the degree to which the PMPU scale assesses the same construct across different languages. The aims of the present study were to (i determine an optimal factor structure for the PMPUQ–SV among university populations using eight versions of the scale (i.e., French, German, Hungarian, English, Finnish, Italian, Polish, and Spanish; and (ii simultaneously examine the measurement invariance (MI of the PMPUQ–SV across all languages. The whole study sample comprised 3038 participants. Descriptive statistics, correlations, and Cronbach’s alpha coefficients were extracted from the demographic and PMPUQ-SV items. Individual and multigroup confirmatory factor analyses alongside MI analyses were conducted. Results showed a similar pattern of PMPU across the translated scales. A three-factor model of the PMPUQ-SV fitted the data well and presented with good psychometric properties. Six languages were validated independently, and five were compared via measurement invariance for future cross-cultural comparisons. The present paper contributes to the assessment of problematic mobile phone use because it is the first study to provide a cross-cultural psychometric analysis of the PMPUQ-SV.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

Science.gov (United States)

Kahlert, H; Cromwell, O; Fiebig, H

2003-09-01

The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

The use of /sup 125/I recombinant DNA/sub 125/ derived human erythropoietin (R-HuEPO) as a replacement for /sup 125/I human urinary epo as tracer antigen in a radioimmunoassay for human epo

International Nuclear Information System (INIS)

Cotes, P.M.; Tam, R.C.; GainesDas, R.E.

1987-01-01

This paper represents evidence that in a radioimmunoassay for human erythropoietin, recombinant DNA derived human erythropoietin can replace highly purified human urinary erythropoietin in the preparation of radioiodinated tracer antigen

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

Science.gov (United States)

Vora, J P; Owens, D R; Dolben, J; Atiea, J A; Dean, J D; Kang, S; Burch, A; Brange, J

1988-11-12

To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. Study in normal people at a diabetes research unit and a university department of medical physics. Seven healthy male volunteers aged 20-39 not receiving any other drugs. After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and

Report of recombinant norovirus GII.g/GII.12 in Beijing, China.

Directory of Open Access Journals (Sweden)

Shaowei Sang

Full Text Available BACKGROUND: Norovirus (NoV has been recognized as the most important cause of nonbacterial acute gastroenteritis affecting all age group people in the world. Genetic recombination is a common occurance in RNA viruses and many recombinant NoV strains have been described since it was first reported in 1997. However, the knowledge of recombinant NoV in China is extremely limited. METHODS: A total of 685 stool specimens were tested for NoV infection from the acute gastroenteritis patients who visited one general hospital in Beijing from April 2009 to November 2011. The virus recombination was identified by constructing phylogenetic trees of two genes, further SimPlot and the maximum chi-square analysis. RESULTS: The overall positive rate was 9.6% (66/685. GII.4 New Orleans 2009 and GII.4 2006b variants were the dominant genotype. Four GII.g/GII.12 and one GII.12/GII.3 recombinant strains were confirmed, and all derived from adult outpatients. The predictive recombination point occurred at the open reading frame (ORF1/ORF2 overlap. CONCLUSIONS: The GII.g ORF1/GII.12ORF2 recombinant has been reported in several countries and it was the first report of this recombinant in China.

Radiative recombination of excitons in amorphous semiconductors

Energy Technology Data Exchange (ETDEWEB)

Singh, Jai [School of Engineering and Logistics, Faculty Technology, B-41, Charles Darwin University, Darwin, NT 0909 (Australia)]. E-mail: jai.singh@cdu.edu.au

2005-04-15

A theory for calculating the radiative lifetime of excitons in amorphous semiconductors is presented. Four possibilities of excitonic radiative recombination are considered and the corresponding rates are derived at thermal equilibrium. The radiative lifetime is calculated from the inverse of the maximum rate for all the four possibilities. Results agree very well with experiments.

Radiative recombination of excitons in amorphous semiconductors

International Nuclear Information System (INIS)

Singh, Jai

2005-01-01

A theory for calculating the radiative lifetime of excitons in amorphous semiconductors is presented. Four possibilities of excitonic radiative recombination are considered and the corresponding rates are derived at thermal equilibrium. The radiative lifetime is calculated from the inverse of the maximum rate for all the four possibilities. Results agree very well with experiments

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

Directory of Open Access Journals (Sweden)

Sanjukta Chakrabarti

2016-06-01

Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

Effektregnskab for projektet Digitales ll – Det svære valg

DEFF Research Database (Denmark)

Mark, Stine

Effektregnskabet for projektet Digitales ll – Det svære valg er udarbejdet af forskningscenteret INCEVIDA, Aalborg Universitet 2012. Rapporten er gennemført i et samarbejde mellem INCEVIDA, KulturarvNord og Bangsbo Museum. Effektregnskabet tager udgangspunkt i en model for oplevelsesøkonomisk...

Characterization of components released by alkali disruption of simian virus 40

DEFF Research Database (Denmark)

Christiansen, Gunna; Landers, T; Griffith, J

1977-01-01

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and ap...

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

Science.gov (United States)

Kempf, Brian J.; Peersen, Olve B.

2016-01-01

ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

Dielectronic recombination rate coefficients to the excited states of CII from CIII

International Nuclear Information System (INIS)

Kato, Takako; Safronova, U.; Ohira, Mituhiko.

1996-02-01

Energy levels, radiative transition probabilities and autoionization rates for CII including 1s 2 2l2l'nl'' (n=2-6, l'≤(n-1)) states were calculated by using multi-configurational Hartree-Fock (Cowan code) method. Autoionizing levels above three thresholds: 1s 2 2s 2 ( 1 S), 1s 2 2s2p( 3 P), 1s 2 2s2p( 1 P) were considered. Branching ratios related to the first threshold and the intensity factor were calculated for satellite lines of CII ion. The dielectronic recombination rate coefficients to the excited states for n=2-6 are calculated with these atomic data. The rate coefficients are fitted to an analytical formula and the fit parameters are given. The values for higher excited states than n=6 are extrapolated and the total dielectronic recombination rate coefficients are derived. The effective recombination rate coefficient for different electron densities are also derived. (author)

Posttranslational modifications in human plasma MBL and human recombinant MBL

DEFF Research Database (Denmark)

Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

2007-01-01

the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

Collisional-radiative model including recombination processes for W27+ ion★

Science.gov (United States)

Murakami, Izumi; Sasaki, Akira; Kato, Daiji; Koike, Fumihiro

2017-10-01

We have constructed a collisional-radiative (CR) model for W27+ ions including 226 configurations with n ≤ 9 and ł ≤ 5 for spectroscopic diagnostics. We newly include recombination processes in the model and this is the first result of extreme ultraviolet spectrum calculated for recombining plasma component. Calculated spectra in 40-70 Å range in ionizing and recombining plasma components show similar 3 strong lines and 1 line weak in recombining plasma component at 45-50 Å and many weak lines at 50-65 Å for both components. Recombination processes do not contribute much to the spectrum at around 60 Å for W27+ ion. Dielectronic satellite lines are also minor contribution to the spectrum of recombining plasma component. Dielectronic recombination (DR) rate coefficient from W28+ to W27+ ions is also calculated with the same atomic data in the CR model. We found that larger set of energy levels including many autoionizing states gave larger DR rate coefficients but our rate agree within factor 6 with other works at electron temperature around 1 keV in which W27+ and W28+ ions are usually observed in plasmas. Contribution to the Topical Issue "Atomic and Molecular Data and their Applications", edited by Gordon W.F. Drake, Jung-Sik Yoon, Daiji Kato, and Grzegorz Karwasz.

Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

Science.gov (United States)

Fang, Jia; Wei, Yudong; Teng, Xin; Zhao, Shanting; Hua, Jinlian

2018-04-01

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs. © 2017 Wiley Periodicals, Inc.

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

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Mauro Tognon

Full Text Available Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

How hot are drosophila hotspots? examining recombination rate variation and associations with nucleotide diversity, divergence, and maternal age in Drosophila pseudoobscura.

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Brenda Manzano-Winkler

Full Text Available Fine scale meiotic recombination maps have uncovered a large amount of variation in crossover rate across the genomes of many species, and such variation in mammalian and yeast genomes is concentrated to <5kb regions of highly elevated recombination rates (10-100x the background rate called "hotspots." Drosophila exhibit substantial recombination rate heterogeneity across their genome, but evidence for these highly-localized hotspots is lacking. We assayed recombination across a 40Kb region of Drosophila pseudoobscura chromosome 2, with one 20kb interval assayed every 5Kb and the adjacent 20kb interval bisected into 10kb pieces. We found that recombination events across the 40kb stretch were relatively evenly distributed across each of the 5kb and 10kb intervals, rather than concentrated in a single 5kb region. This, in combination with other recent work, indicates that the recombination landscape of Drosophila may differ from the punctate recombination pattern observed in many mammals and yeast. Additionally, we found no correlation of average pairwise nucleotide diversity and divergence with recombination rate across the 20kb intervals, nor any effect of maternal age in weeks on recombination rate in our sample.

Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

NARCIS (Netherlands)

Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

1998-01-01

The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

Derivation methods for clearance levels applied to reactors

International Nuclear Information System (INIS)

Okoshi, Minoru; Seki, Takeo

2001-01-01

In order to support the discussion by the Nuclear Safety Commission, JAERI derived the unconditional clearance levels for concrete and metal arising from the operation and dismantling of nuclear reactors. The clearance levels of 20 radionuclides were derived from 10 μSv/y of individual doses by deterministic approach. In this approach, calculation models were established to assess individual doses resulting from 73 exposure pathways related to disposal and recycle/reuse, and realistic parameter values were selected considering Japanese natural and social conditions. The appropriateness of selected parameter values was confirmed by stochastic analyses. (author)

Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults.

Science.gov (United States)

Brown, Bruce K; Cox, Josephine; Gillis, Anita; VanCott, Thomas C; Marovich, Mary; Milazzo, Mark; Antonille, Tanya Santelli; Wieczorek, Lindsay; McKee, Kelly T; Metcalfe, Karen; Mallory, Raburn M; Birx, Deborah; Polonis, Victoria R; Robb, Merlin L

2010-11-05

The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. ClinicalTrials.gov NCT00057525.

Immunoglobulin class-switch recombination deficiencies.

Science.gov (United States)

Durandy, Anne; Kracker, Sven

2012-07-30

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

The suitability of 129SvEv mice for studying depressive-like behaviour: both males and females develop learned helplessness.

Science.gov (United States)

Chourbaji, Sabine; Pfeiffer, Natascha; Dormann, Christof; Brandwein, Christiane; Fradley, Rosa; Sheardown, Malcolm; Gass, P

2010-07-29

Behavioural studies using transgenic techniques in mice usually require extensive backcrossing to a defined background strain, e.g. to C57BL/6. In this study we investigated whether backcrossing can be replaced by using the 129SvEv strain from which the embryonic stem cells are generally obtained for gene targeting strategies to analyze e.g. depression-like behaviour. For that purpose we subjected male and female 129SvEv mice to two frequently used depression tests and compared them with commonly used C57BL/6 mice. 129SvEv and C57BL/6 mice exhibited differing profiles with regard to locomotion and pain sensitivity. However, in the learned helplessness paradigm, a procedure, which represents a valid method to detect depressive-like behaviour, 129SvEv animals develop a similar level of helplessness as C57BL/6 mice. One great advantage of the 129SvEv animals though, is the fact that in this strain even females develop helplessness, which could not be produced in C57BL/6 mice. In the tail suspension test, both genders of 129SvEv exhibited more despair behaviour than C57BL/6 animals. We therefore suggest that this strain may be utilized in the establishment of new test procedures for affective diseases, since costly and time-consuming backcrossing can be prevented, depressive-like behaviour may be analyzed effectively, and gender-specific topics could be addressed in an adequate way. Copyright 2010 Elsevier B.V. All rights reserved.

Analysis of intermolecular RNA-RNA recombination by rubella virus

International Nuclear Information System (INIS)

Adams, Sandra D.; Tzeng, W.-P.; Chen, M.-H.; Frey, Teryl K.

2003-01-01

To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating

Formation of pyrimidine dimers in Simian virus 40 chromosomes and DNA in vitro: effects of salt

International Nuclear Information System (INIS)

Edenberg, H.J.

1984-01-01

Simian virus 40 chromosomes were used to determine whether packaging of DNA into chromatin affected the yield of cylcobutane pyrimidine dimers introduced by ultraviolet light (254 nm). SV40 chromatin and purified SV40 DNA (radioactively labeled with different isotopes) were mixed and irradiated in vitro. The proteins were extracted and pyrimidine dimers detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. When irradiation was carried out in the presence of at least 0.05 M NaCl the same number of dimers were formed in chromatin as in free DNA. Irradiation in the absence of NaCl, however, reduced the relative yield of dimers in chromatin to 89% of that in free DNA. Different methods of chromatin preparation did not influence these results. (author)

Evaluation of the 40 K internal exposure on the inhabitants of Rio de Janeiro city, Brazil

International Nuclear Information System (INIS)

Santos, Eliane E.; Lauria, Dejanira C.; Amaral, Eliana C.S.; Carvalho, Laercio L.

2002-01-01

Among the naturally occurring radionuclides 40 K is the highest contributor for the intake dose. The aiming of this research was to determine the activity concentrations of 40 K in the main consumed foodstuffs by the adult inhabitants of Rio de Janeiro city in view of estimating its daily intake and arisen intake dose. The 40 K activity concentrations in the foodstuffs ranged from 20 to 544 Bq/kg fresh and the estimated daily intake was 61 Bq. The highest contribution for its intake arises from consumption of bean (35%), beef (11%), potato (8%) and rice (8%). The total annual effective dose was estimated to be 138 mSv/a. Value that falls into the range of international literature reported value (from 122 to 200 μSv/a. (author)

Equilibrium Investment Strategy for DC Pension Plan with Inflation and Stochastic Income under Heston’s SV Model

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Jingyun Sun

2016-01-01

Full Text Available We consider a portfolio selection problem for a defined contribution (DC pension plan under the mean-variance criteria. We take into account the inflation risk and assume that the salary income process of the pension plan member is stochastic. Furthermore, the financial market consists of a risk-free asset, an inflation-linked bond, and a risky asset with Heston’s stochastic volatility (SV. Under the framework of game theory, we derive two extended Hamilton-Jacobi-Bellman (HJB equations systems and give the corresponding verification theorems in both the periods of accumulation and distribution of the DC pension plan. The explicit expressions of the equilibrium investment strategies, corresponding equilibrium value functions, and the efficient frontiers are also obtained. Finally, some numerical simulations and sensitivity analysis are presented to verify our theoretical results.

Epilepsy caused by an abnormal alternative splicing with dosage effect of the SV2A gene in a chicken model.

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Marine Douaud

Full Text Available Photosensitive reflex epilepsy is caused by the combination of an individual's enhanced sensitivity with relevant light stimuli, such as stroboscopic lights or video games. This is the most common reflex epilepsy in humans; it is characterized by the photoparoxysmal response, which is an abnormal electroencephalographic reaction, and seizures triggered by intermittent light stimulation. Here, by using genetic mapping, sequencing and functional analyses, we report that a mutation in the acceptor site of the second intron of SV2A (the gene encoding synaptic vesicle glycoprotein 2A is causing photosensitive reflex epilepsy in a unique vertebrate model, the Fepi chicken strain, a spontaneous model where the neurological disorder is inherited as an autosomal recessive mutation. This mutation causes an aberrant splicing event and significantly reduces the level of SV2A mRNA in homozygous carriers. Levetiracetam, a second generation antiepileptic drug, is known to bind SV2A, and SV2A knock-out mice develop seizures soon after birth and usually die within three weeks. The Fepi chicken survives to adulthood and responds to levetiracetam, suggesting that the low-level expression of SV2A in these animals is sufficient to allow survival, but does not protect against seizures. Thus, the Fepi chicken model shows that the role of the SV2A pathway in the brain is conserved between birds and mammals, in spite of a large phylogenetic distance. The Fepi model appears particularly useful for further studies of physiopathology of reflex epilepsy, in comparison with induced models of epilepsy in rodents. Consequently, SV2A is a very attractive candidate gene for analysis in the context of both mono- and polygenic generalized epilepsies in humans.

Electrochemical cleaning of Sv-08G2S wire surface

International Nuclear Information System (INIS)

Kozlov, E.I.; Degtyarev, V.G.; Novikov, M.P.

1981-01-01

Results of industrial tests of the Sv-08G2S wire with different state of surface fwith technological lubrication, after mechanical cleaning, with electrochemically cleaned surface) are presented. Advantages of welding-technological properties of the wire with electroe chemically cleaned surface are shown. An operation principle of the electrochemical cleaning facility is described. A brief specf ification f of the facility is given [ru

DNA degradation and reduced recombination following UV irradiation during meiosis in yeast (Saccharomyces cerevisiae)

International Nuclear Information System (INIS)

Salts, Y.; Pinon, R.; Simchen, G.

1976-01-01

Irradiation of meiotic yeast cells with moderate doses of ultraviolet irradiation (1,600 erg/mm 2 ) leads to the arrest of premeiotic DNA synthesis, massive (5-40%) DNA degradation, and a 40-50% loss of cell viability. In contrast, such doses of UV irradiation had a minor effect on viability (15-20% loss) of logarithmically growing cells, and no comparable DNA degradation was observed in irradiated synchronized vegetative cells. Meiotic recombination is also affected by UV irradiation. When administered at a stage comparable to meiotic prophase, low doses of irradiation result in a reduction in recombination frequency without significantly affecting cell viability. (orig.) [de

Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications.

Science.gov (United States)

Korotkova, Ekaterina; Laassri, Majid; Zagorodnyaya, Tatiana; Petrovskaya, Svetlana; Rodionova, Elvira; Cherkasova, Elena; Gmyl, Anatoly; Ivanova, Olga E; Eremeeva, Tatyana P; Lipskaya, Galina Y; Agol, Vadim I; Chumakov, Konstantin

2017-11-22

Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing) of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These "weak" segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Science.gov (United States)

Macao, Bertil; Hoyer, Wolfgang; Sandberg, Anders; Brorsson, Ann-Christin; Dobson, Christopher M; Härd, Torleif

2008-01-01

Background Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation. Results Coexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40) and Aβ(1–42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Aβ(1–42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. Conclusion The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Aβ(1–42) is reported. PMID:18973685

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Directory of Open Access Journals (Sweden)

Dobson Christopher M

2008-10-01

Full Text Available Abstract Background Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD. The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation. Results Coexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40 and Aβ(1–42, yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42 carrying the Arctic (E22G mutation, which causes early onset familial AD. Aβ(1–42E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. Conclusion The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G of Aβ(1–42 is reported.

Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System.

Science.gov (United States)

An, Mahru C; O'Brien, Robert N; Zhang, Ningzhe; Patra, Biranchi N; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M

2014-04-15

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

Science.gov (United States)

Rozov, S M; Permyakova, N V; Deineko, E V

2018-03-01

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

Epidermal cell-shape regulation and subpopulation kinetics during butyrate-induced terminal maturation of normal and SV40-transformed human keratinocytes: epithelial models of differentiation therapy.

Science.gov (United States)

Staiano-Coico, L; Steinberg, M; Higgins, P J

1990-10-15

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.

Recombinant methioninase effectively targets a Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) nude-mouse model.

Science.gov (United States)

Murakami, Takashi; Li, Shukuan; Han, Qinghong; Tan, Yuying; Kiyuna, Tasuku; Igarashi, Kentaro; Kawaguchi, Kei; Hwang, Ho Kyoung; Miyake, Kentaro; Singh, Arun S; Nelson, Scott D; Dry, Sarah M; Li, Yunfeng; Hiroshima, Yukihiko; Lwin, Thinzar M; DeLong, Jonathan C; Chishima, Takashi; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Eilber, Fritz C; Hoffman, Robert M

2017-05-30

Methionine dependence is due to the overuse of methionine for aberrant transmethylation reactions in cancer. Methionine dependence may be the only general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]). The cloned methioninase, termed recombinant methioninase, or rMETase, has been tested in mouse models of human cancer cell lines. Ewing's sarcoma is recalcitrant disease even though development of multimodal therapy has improved patients'outcome. Here we report efficacy of rMETase against Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. Eight Ewing's sarcoma PDOX mice were randomized into untreated control group (n = 4) and rMETase treatment group (n = 4). rMETase (100 units) was injected intraperitoneally (i.p.) every 24 hours for 14 consecutive days. All mice were sacrificed on day-15, 24 hours after the last rMETase administration. rMETase effectively reduced tumor growth compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group. Body weight did not significantly differ at any time points between the 2 groups. The present study is the first demonstrating rMETase efficacy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as Ewing's sarcoma.

Časopis "Módní svět" a "Lada" v kulturním kontextu

OpenAIRE

Šrolová, Kristýna

2010-01-01

The thesis discusses tbe fashion magazine Módní svět (Fashion World) and its enclosure Lada, which it implants into their cultural and historica1 context. The magazine Módní svět (Fashion World) was puhli,hed from 1875 to 1935 and thi, a1,o deline, lhe time circmnscription of tbe themes in tbe thesis. It contextua11y deals with tbe women society during tbe tum of 19th to 20th century, with women's emancipatory movement, with tbe beginning of publishing of rn.agazines for women and fashion mag...

A high-quality narrow passband filter for elastic SV waves via aligned parallel separated thin polymethylmethacrylate plates

OpenAIRE

Jun Zhang; Yaolu Liu; Wensheng Yan; Ning Hu

2017-01-01

We designed a high-quality filter that consists of aligned parallel polymethylmethacrylate (PMMA) thin plates with small gaps for elastic SV waves propagate in metals. Both the theoretical model and the full numerical simulation show the transmission spectrum of the elastic SV waves through such a filter has several sharp peaks with flawless transmission within the investigated frequencies. These peaks can be readily tuned by manipulating the geometry parameters of the PMMA plates. Our invest...

Studies on reduction coefficients of various buildings of radiation derived from Fukushima Daiichi nuclear power plant accident

International Nuclear Information System (INIS)

Izumi, Yukio; Chida, Tooru; Kato, Yoshiharu

2012-01-01

Reduction coefficients (RC) were studied by actual measurement of radiation 1 year after the Accident in the title to re-evaluate the RC 0.40 value of wooden houses (IAEA), also defined by Ministries of Education, Culture, Sports, Science and Technology (MECSST) and of Environment (ME). Dose rates were measured with HORIBA's PA-1000 Radi with the detector of CsI(Tl) having the effective range of 0.001-9.999 mcSv/h, in Ibaraki and Fukushima prefectures, of wooden houses, various public buildings of light steel frame and/or reinforced concrete like general apartment, meeting places, community centers, big shops, hotels, etc. Measurement was performed at 0.5 m height from the floor of living places and grounds, and at 1 m height for public spaces, from Jan. to Mar., 2012. Outdoor dose rates including the background values were found higher around Fukushima Station (0.75-1.56 mcSv/h) than Ibaraki Station (0.09-0.25). RC of wooden houses in Ibaraki prefecture were found to be the average 0.68 (n=86), of light steel frame buildings, 0.60 (8) and of reinforced concrete ones, 0.71 (26), which were all insignificant between. In Fukushima City, dose rates in reinforced concrete buildings were obviously lower than outdoor with RC 0.09-0.12 (n=22). RC of wooden houses was 0.51 (3) and of light frame ones, 0.14 (1). Above observed RC =0.68 of wooden houses was 1.7 times as high as the guideline (GL) 0.40; 0.71 of reinforced concrete houses, 3.5 times (GL 0.20); and/or 0.10 of multi-floor buildings, 2 or 10 times (GL 0.05 or 0.01). Results showed that observed RC were all higher than the disaster preventive GL values, possibly leading to underestimation of exposed dose. In addition, should be re-considered such limits as the annually accumulated dose 20 mSv and ambient dose rate 3.8 mcSv/h for schoolchildren defined by MECSST, and the additional dose 1 mSv and 0.23 mcSv/h for indicating the selective area to be surveyed for contamination by ME, all values of which were defined

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

International Nuclear Information System (INIS)

Funk, W.D.; MacGillivray, R.T.A.; Mason, A.B.; Brown, S.A.; Woodworth, R.C.

1990-01-01

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO 4 -polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19 F-Tyr hTF/2N gave four well-resolved 19 F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Science.gov (United States)

Yan, Fang; Cao, Hanwei; Cover, Timothy L.; Washington, M. Kay; Shi, Yan; Liu, LinShu; Chaturvedi, Rupesh; Peek, Richard M.; Wilson, Keith T.; Polk, D. Brent

2011-01-01

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria–derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40’s effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation. PMID:21606592

Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding

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Robert Gustafsson

2018-04-01

Full Text Available Botulinum neurotoxins (BoNTs are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G. Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C as their protein receptors. We here present a high resolution (2.0 Å co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.

Dielectronic recombination rate coefficients to the excited states of CI from CII

International Nuclear Information System (INIS)

Dubau, J.; Kato, T.; Safronova, U.I.

1998-01-01

The dielectronic recombination rate coefficients to the excited states for n=2-6 are calculated including 1s 2 2l 1 2l 2 2l 3 nl (n=2-6, l≤(n-1)) states. The values for the excited states higher than n=6 are extrapolated and the total dielectronic recombination rate coefficients are derived. The rate coefficients to the excited states are fitted to an analytical formula and the fit parameters are given. (author)

Recombiner

International Nuclear Information System (INIS)

Osumi, Morimichi.

1979-01-01

Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

Suitability of digital elevation models generated by uav photogrammetry for slope stability assessment (case study of landslide in Svätý Anton, Slovakia

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Rusnák Miloš

2016-12-01

Full Text Available Assessing the accuracy of photogrammetrically-derived digital elevation models (DEMs from UAV is essential in many geoscience disciplines. The suitability of different DEM devised for slope stability assessment was evaluated in the example of the landslide in Svätý Anton village in Slovakia. Aerial data was acquired during a one-day field campaign in autumn 2014. The point cloud from 218 images (54,607,748 points was manually classified into 7 different classes for filtering vegetation cover and buildings. Assessment of vertical differences between the UAV derived elevation model and real terrain surface was based on comparison of control points targeted by GPS (337 points and unclassified and ground classified point cloud for raster elevation models with 1, 5, 10, 20 and 50 cm pixel resolution.

A physical model study of the travel times and conversion point locations of P-SV converted waves in vertical transversely isotropic media

Science.gov (United States)

Tseng, C.

2013-12-01

In exploration seismology, subsurface medium commonly exhibits anisotropy, characterized by a vertical transversely isotropic (VTI) model. Due to the need of exploring small reservoirs in complex structures, the seismic exploration is extended to deal with anisotropic media. The P-S converted wave seismic exploration is a relatively inexpensive, broadly applicable, and effective way to obtain the S-wave information of the medium. In anisotropic traveltime analysis, the moveout curve of horizontal P-SV event can help to determine the ratio of the P- and SV-wave vertical velocities, the normal moveout (NMO) velocity of SV-waves, and the anisotropy parameters. The P-SV conversion point (CP) location is of great importance to P-SV data binning, NMO corrections and common conversion point (CCP) stacking, and the anisotropy has a more significant effect on the conversion point location than on the moveout. In this study, we attempt to inspect the theoretical non-hyperbolic moveout and CP equations for the P-SV waves reflected from a VTI layer by numerical calculations and physical modeling. We are also interested in visualizing the variations of the conversion point locations from a designed VTI medium. In traveltime analysis, the theoretical moveout curve is accurate up to offsets about one and a half times the reflector depth (x/z=1.5). However, the moveout curve computed by Fermat's principle fits well to the physical data. The CP locations of P-SV waves are similar to those calculated by Fermat's principle and theoretical CP equation, which are verified by the physical modeling.

Evidence of recombination in intrapatient populations of hepatitis C virus.

Science.gov (United States)

Sentandreu, Vicente; Jiménez-Hernández, Nuria; Torres-Puente, Manuela; Bracho, María Alma; Valero, Ana; Gosalbes, María José; Ortega, Enrique; Moya, Andrés; González-Candelas, Fernando

2008-09-18

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially

Overexpressed Calponin3 by Subsonic Vibration Induces Neural Differentiation of hUC-MSCs by Regulating the Ionotropic Glutamate Receptor.

Science.gov (United States)

Kim, Hyun-Jung; Kim, Jin-Hee; Song, Yeo-Ju; Seo, Young-Kwon; Park, Jung-Keug; Kim, Chan-Wha

2015-09-01

In this study, we used proteomics to investigate the effects of sonic vibration (SV) on mesenchymal stem cells derived from human umbilical cords (hUC-MSCs) during neural differentiation to understand how SV enhances neural differentiation of hUC-MSCs. We investigated the levels of gene and protein related to neural differentiation after 3 or 5 days in a group treated with 40-Hz SV. In addition, protein expression patterns were compared between the control and the 40-Hz SV-treated hUC-MSC groups via a proteomic approach. Among these proteins, calponin3 (CNN3) was confirmed to have 299 % higher expression in the 40-Hz SV stimulated hUC-MSCs group than that in the control by Western blotting. Notably, overexpression of CNN3-GFP in Chinese hamster ovary (CHO)-K1 cells had positive effects on the stability and reorganization of F-actin compared with that in GFP-transfected cells. Moreover, CNN3 changed the morphology of the cells by making a neurite-like form. After being subjected to SV, messenger RNA (mRNA) levels of glutamate receptors such as PSD95, GluR1, and NR1 as well as intracellular calcium levels were upregulated. These results suggest that the activity of glutamate receptors increased because of CNN3 characteristics. Taken together, these results demonstrate that overexpressed CNN3 during SV increases expression of glutamate receptors and promotes functional neural differentiation of hUC-MSCs.

Effects of Bimolecular Recombination on Impedance Spectra in Organic Semiconductors: Analytical Approach.

Science.gov (United States)

Takata, Masashi; Takagi, Kenichiro; Nagase, Takashi; Kobayashi, Takashi; Naito, Hiroyoshi

2016-04-01

An analytical expression for impedance spectra in the case of double injection (both electrons and holes are injected into an organic semiconductor thin film) has been derived from the basic transport equations (the current density equation, the continuity equation and the Possion's equation). Capacitance-frequency characteristics calculated from the analytical expression have been examined at different recombination constants and different values of mobility balance defined by a ratio of electron mobility to hole mobility. Negative capacitance appears when the recombination constant is lower than the Langevin recombination constant and when the value of the mobility balance approaches unity. These results are consistent with the numerical results obtained by a device simulator (Atlas, Silvaco).

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

Science.gov (United States)

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line; Delpeyroux, Francis

2014-08-05

Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges

Når det er svært at være ung i DK

DEFF Research Database (Denmark)

Nielsen, Jens Christian; Sørensen, Niels Ulrik; Grubb, Ane

I rapporten Når det er svært at være ung i DK – unges beretninger om mistrivsel og ungdomsliv præsenteres resultaterne af et kvalitativt studie af mistrivsel og ungdomsliv blandt 15-24-årige unge i Danmark. Studiet bygger på dybdegående interviews med 33 unge fra forskellige dele af landet, der...... fortæller om deres erfaringer med diverse mistrivselsformer, som ensomhed, selvskadende adfærd og mobning, og om deres besvær med at håndtere de krav, udfordringer og muligheder, der i øvrigt præger det moderne ungdomsliv. Studiet indgår i det treårige forskningsprojekt Når det er svært at være ung i DK...

Fast Dissemination of New HIV-1 CRF02/A1 Recombinants in Pakistan.

Directory of Open Access Journals (Sweden)

Yue Chen

Full Text Available A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%, together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15% but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a while 12 (38% were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan.

Fast Dissemination of New HIV-1 CRF02/A1 Recombinants in Pakistan

Science.gov (United States)

Chen, Yue; Hora, Bhavna; DeMarco, Todd; Shah, Sharaf Ali; Ahmed, Manzoor; Sanchez, Ana M.; Su, Chang; Carter, Meredith; Stone, Mars; Hasan, Rumina; Hasan, Zahra; Busch, Michael P.; Denny, Thomas N.; Gao, Feng

2016-01-01

A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan. PMID:27973597

Dielectronic recombination rate coefficients to the excited states of CI from CII

Energy Technology Data Exchange (ETDEWEB)

Dubau, J. [Observatoire of Paris, 92 MEUDON (France); Kato, T.; Safronova, U.I.

1998-01-01

The dielectronic recombination rate coefficients to the excited states for n=2-6 are calculated including 1s{sup 2}2l{sub 1}2l{sub 2}2l{sub 3}nl (n=2-6, l{<=}(n-1)) states. The values for the excited states higher than n=6 are extrapolated and the total dielectronic recombination rate coefficients are derived. The rate coefficients to the excited states are fitted to an analytical formula and the fit parameters are given. (author)

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support.

Science.gov (United States)

Giri, Shibashish; Bader, Augustinus

2014-09-01

Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a

Analysis of relation between the mutation frequencies and somatic recombination induced by neutrons and the age of D. Melanogaster larvae

International Nuclear Information System (INIS)

Guzman R, J.; Zambrano A, F.; Paredes G, L.; Delfin L, A.; Quiroz R, C.

1998-01-01

Neutrons are subatomic particles with neutral electric charge, equal zero, which are emitted during the fissile material fission in nuclear reactors. It is known a little about biological effects induced by neutrons. There is a world interest in the use of reactors and accelerators for patients radiotherapy using neutrons with the purpose to destroy malignant cells of deep tumours where traditional methods have not given satisfactory results. There for it is required to do wide studies of biological effects of neutrons as well as their dosimetry. It was used the Smart test (Somatic Mutation and Recombination Test) of D. Melanogaster for quantifying the mutation induction and somatic recombination induced by neutrons of the National Institute of Nuclear Research reactor, at power of 300 and 1000 k W, with equivalent doses calculated 95.14 and 190.2 Sv for 300 k W and of 25.64 and 51.29 Sv for 1000 k W, using larvae with 72 or 96 hours aged. It was observed a linear relation between equivalent dose and genetic effects frequency, these last were greater when the reactor power was 1000 k W than those 300 k W. It was observed too that the damage was greater in 96 hours larvae than those 72 hours. The stain size presented an inverse relation with respect to larvae age. It is concluded that the Smart system is sensitive to neutrons effect and it responds of a directly proportional form to radiation dose, as well as to dose rate. It is noted more the effect when are used larvas in pre pupa stage where the irradiation target (imagal cells) is greater. The Smart is sensitive to damage induced by neutrons , thus can be used to studying its direct biological effects or by the use of chemical modulators. (Author)

Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

Energy Technology Data Exchange (ETDEWEB)

Lau, Aik Jiang; Chang, Thomas K.H., E-mail: thomas.chang@ubc.ca

2014-06-01

The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate hCAR-SV

Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications

Directory of Open Access Journals (Sweden)

Ekaterina Korotkova

2017-11-01

Full Text Available Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

The content of 226Ra, 210Pb, 210Po and 40K on tobacco of some cigarette brands were sold in Bandung, West Java

International Nuclear Information System (INIS)

Putu Sukmabuana

2016-01-01

This study on the measurements of 226 Ra, 210 Pb, 210 Po and 40 K natural radionuclides contained in tobacco smoke, in order to estimate the effective dose received by smoker has been carried out. The samples have been measured covering 14 brands of cigarettes are commonly sold and consumed in Indonesia. After the tobacco is dried and mashed, the concentration of 226 Ra, 210 Pb and 40 K counted using γ-ray spectrometry, for 80,000 seconds. It was obtained that the average concentration of 226 Ra was 4.18 ± 0.67 Bq/kg, for 210 Pb was 4.71 ± 0.82 Bq/kg, while the average concentration of 40 K was 26.50 ± 2.08 Bq/kg. 210 Po calculated based on radioactive equilibrium, the result was 4.09 ± 0.71 Bq/kg. By using a dose coefficients given by ICRP, the estimated annual effective dose received by the smoker was calculated. The average annual effective dose for 226 Ra was 80.46 ± 12.93 µSv/year, for 210 Pb was 28.47 ± 4.96 µSv/year, and 210 Po was 74.31 ± 12.96 µSv/year. Estimated effective dose for 40 K radionuclide can not be done because the dose coefficients for 40 K is not available in ICRP 71. By summing the average dose of 226 Ra, 210 Pb and 210 Po, obtained total effective dose estimated annual average was 183.24 µSv/year or around 14,5 % of the dose limit for radiation exposure by inhalation in the world (1260 µSv/year). In summary the effective dose above is still relatively small, but nevertheless when radioactive substances and chemicals in cigarette smoke enters the bloodstream it can affect the entire body. This is why smoking causes so many diseases, including cancer, heart disease and various lung diseases. (author)

Quinazolin-4-one derivatives

DEFF Research Database (Denmark)

Mosley, Cara A; Acker, Timothy M; Hansen, Kasper Bø

2010-01-01

We describe a new class of subunit-selective antagonists of N-methyl D-aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives...

Når det er svært at være ung i DK

DEFF Research Database (Denmark)

Nielsen, Jens Christian; Sørensen, Niels Ulrik

Når det er svært at være ung i DK – viden og råd om unges trivsel og mistrivsel er afslutningen på et større forskningsprojekt om unges trivsel og mistrivsel. Hæftet præsenterer forskningsprojektets hovedkonklusioner og giver desuden en række råd og ideer til, hvordan voksne kan hjælpe unge med...... at håndtere mistrivsel. Hæftet er udarbejdet af forskerne Jens Christian Nielsen og Niels Ulrik Sørensen fra Center for Ungdomsforskning. Det er den sidste publikation i forskningsprojektet Når det er svært at være ung i DK, der fra 2008-2011 har belyst unges trivsel og mistrivsel. Projektet bygger både på en...

Når det er svært at være ung i DK

DEFF Research Database (Denmark)

Nielsen, Jens Christian; Sørensen, Niels Ulrik; Ozmec, Martha Nina

I rapporten Når det er svært at være ung i DK – unges trivsel og mistrivsel i tal offentliggør Center for Ungdomsforskning resultaterne af en stor videnskabelig spørgeskemaundersøgelse om trivsel og mistrivsel blandt 15-24-årige unge i Danmark. Rapporten indgår i det treårige forskningsprojekt Når...... det er svært at være ung i DK, som Center for Ungdomsforskning udfører med støtte fra Egmont Fonden. Rapporten bygger på telefoninterviews med 3.481 unge, der udgør et repræsentativt udsnit af alle 15-24-årige unge i Danmark. I rapporten forfølges de unges trivsel og mistrivsel gennem to spor: 1) Et...

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

Science.gov (United States)

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Detailed modelling of processes inside a catalytic recombiner for hydrogen removal

International Nuclear Information System (INIS)

Heitsch, M.

1999-01-01

Under accidental conditions, considerable amounts of hydrogen may be released into the containment. Catalytic reacting surfaces in recombiners are a reliable method to recombine this hydrogen and other burnable gases like carbon monoxide from the atmosphere in a passive way. Many experiments have been carried out to study the main phenomena occurring inside recombiners, like the efficiency of hydrogen removal, the start-up conditions, poisoning, oxygen starvation, steam and water impact, and others. In addition, the global behavior of a given recombiner device in a larger environment has been investigated in order to demonstrate the effectiveness and to facilitate the derivation of simplified models for long term, severe accident analyses. These long-term severe accident models are complemented by detailed investigations to understand the interaction of chemistry and flow inside a recombiner box. This helps to provide the dependencies of non-measurable variables (e.g. the reaction rate distribution), of local surface temperatures etc. to make long-term or system models more reliable. It also offers possibilities for increasing the chemical efficiency by optimising the geometric design properly. Computational Fluid Dynamics (CFD) codes are available for use as development tools to include the specifics of catalytic surface reactors. The present paper describes the use of the code system CFX [1] for creating a recombiner model. Some model predictions are compared to existing test data. (author)

Genome-wide recombination rate variation in a recombination map of cotton.

Science.gov (United States)

Shen, Chao; Li, Ximei; Zhang, Ruiting; Lin, Zhongxu

2017-01-01

Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species' genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton.

Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

Science.gov (United States)

Stukenbrock, Eva H; Dutheil, Julien Y

2018-03-01

Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers.

Science.gov (United States)

Camargo, O A; Souza, E A; Mendes-Costa, M C; Santos, J B; Soares, M A

2007-09-30

We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively.

Recirculation in the Fram Strait and transports of water in and north of the Fram Strait derived from CTD data

Directory of Open Access Journals (Sweden)

M. Marnela

2013-05-01

Full Text Available The volume, heat and freshwater transports in the Fram Strait are estimated from geostrophic computations based on summer hydrographic data from 1984, 1997, 2002 and 2004. In these years, in addition to the usually sampled section along 79° N, a section between Greenland and Svalbard was sampled further north. Quasi-closed boxes bounded by the two sections and Greenland and Svalbard can then be formed. Applying conservation constraints on these boxes provides barotropic reference velocities. The net volume flux is southward and varies between 2 and 4 Sv. The recirculation of Atlantic water is about 2 Sv. Heat is lost to the atmosphere and the heat loss from the area between the sections averaged over the four years is about 10 TW. The net heat (temperature transport is 20 TW northward into the Arctic Ocean, with large interannual differences. The mean net freshwater added between the sections is 40 mSv and the mean freshwater transport southward across 79° N is less than 60 mSv, indicating that most of the liquid freshwater leaving the Arctic Ocean through Fram Strait in summer is derived from sea ice melt in the northern vicinity of the strait. In 1997, 2001 and 2003 meridional sections along 0° longitude were sampled and in 2003 two smaller boxes can be formed, and the recirculation of Atlantic water in the strait is estimated by geostrophic computations and continuity constraints. The recirculation is weaker close to 80° N than close to 78° N, indicating that the recirculation is mainly confined to the south of 80° N. This is supported by the observations in 1997 and 2001, when only the northern part of the meridional section, from 79° N to 80° N, can be computed with the constraints applied. The recirculation is found strongest close to 79° N.

Engineered Modular Recombinant Transporters: Application of New Platform for Targeted Radiotherapeutic Agents to α-Particle Emitting 211At

International Nuclear Information System (INIS)

Rosenkranz, Andrey A.; Vaidyanathan, Ganesan; Pozzi, Oscar R.; Lunin, Vladimir G.; Zalutsky, Michael R.; Sobolev, Alexander S.

2008-01-01

Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). Methods and Materials: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[ 211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. Results: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [ 211 At]astatide for all three cell lines. Conclusion: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted α-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor

Screening of recombinant inbred lines for salinity tolerance in bread ...

African Journals Online (AJOL)

Screening a large number of plants for salinity tolerance is not easy, therefore this investigation was performed to evaluate and screen 186 F8 recombinant inbred lines (RILs) derived from a cross between Superhead#2 (Super Seri) and Roshan wheat varieties for salinity tolerance. All the individuals were evaluated under ...

Optimal recombination in genetic algorithms for combinatorial optimization problems: Part II

Directory of Open Access Journals (Sweden)

Eremeev Anton V.

2014-01-01

Full Text Available This paper surveys results on complexity of the optimal recombination problem (ORP, which consists in finding the best possible offspring as a result of a recombination operator in a genetic algorithm, given two parent solutions. In Part II, we consider the computational complexity of ORPs arising in genetic algorithms for problems on permutations: the Travelling Salesman Problem, the Shortest Hamilton Path Problem and the Makespan Minimization on Single Machine and some other related problems. The analysis indicates that the corresponding ORPs are NP-hard, but solvable by faster algorithms, compared to the problems they are derived from.

Initial recombination of ions in gases (Air). Anfangsrekombination von Ionen in gasen (Luft)

Energy Technology Data Exchange (ETDEWEB)

Booz, J.; Ebert, H. G.

1962-08-15

The dependence of the initial recombination coefficients on the ion density, the ionization length, and the recombination time was theoretically and experimentally investigated for the case of ionization of air with x rays. The experiments were carried out at initial ion densities between 5 x 10/sup 4/ and 2 x 10/sup 6/ ion pairs/cm/sup 3/ and at ionization lengths between 100 mu sec and 3 msec. There was satisfactory agreement with the theoretically derived formula. (tr-auth)

Sigma Virus (DMelSV Incidence in Lines of Drosophila melanogaster Selected for Survival following Infection with Bacillus cereus

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Meghan L. Bentz

2017-01-01

Full Text Available The immune response of Drosophila melanogaster is complex and involves both specific and general responses to parasites. In this study we tested for cross-immunity for bacteria and viruses by scoring the incidence of infection with the vertically transmitted Sigma virus (DMelSV in the progeny of a cross between females transmitting DMelSV at high frequencies and males from lines subjected to three selection regimes related to resistance to Bacillus cereus. There was no significant difference in transmission of DMelSV among selection regimes, though results suggest that the B. cereus selected lines had lower rates of infection by DMelSV. We found a significant difference in viral infection with respect to the sex of the progeny, with males consistently less likely to be infected than females. Given a finite energy budget, flies that have experienced immune system challenge may show alterations in other life history traits. Later eclosing progeny were also less likely to be infected than earlier eclosing progeny, indicating a relationship with development time. Finally, there was a significant interaction between the timing of collection and the sex of the progeny, such that later eclosing males were the most resistant group. Increased development time is sometimes associated with increased energy acquisition; from this perspective, increased development time may be associated with acquiring sufficient resources for effective resistance.

Dielectronic recombination rate coefficients to excited states of Be-like oxygen

Energy Technology Data Exchange (ETDEWEB)

Murakami, Izumi; Safronova, Ulyana I.; Kato, Takako

2001-05-01

We have calculated energy levels, radiative transition probabilities, and autoionization rates for Be-like oxygen (O{sup 4+}) including ls{sup 2}2lnl' (n=2 - 8, l {<=} n - 1) and 1s{sup 2}3l'nl (n=3 - 6, l {<=} n - l) states by multi-configurational Hartree-Fock method (Cowan code) and perturbation theory Z-expansion method (MZ code). The state selective dielectronic recombination rate coefficients to excited states of Be-like O ions are obtained. Configuration mixing plays an important role for the principal quantum number n distribution of the dielectronic recombination rate coefficients for 2snl (n {<=} 5) levels at low electron temperature. The orbital angular momentum quantum number l distribution of the rate coefficients shows a peak at l = 4. The total dielectronic recombination rate coefficient is derived as a function of electron temperature. (author)

Reply to Comments on Measuring marine iron(III) complexes by CLE-AdSV

NARCIS (Netherlands)

Town, R.M.; Leeuwen, van H.P.

2005-01-01

The interpretation of CLE-AdSV based iron(iii) speciation data for marine waters has been called into question in light of the kinetic features of the measurement. The implications of the re-think may have consequences for understanding iron biogeochemistry and its impact on ecosystem functioning.

Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma-derived FVIII or FIX concentrate: results from the French haemophilia cohort.

Science.gov (United States)

Gaboulaud, Valérie; Parquet, Armelle; Tahiri, Cedric; Claeyssens, Ségolène; Potard, Valérie; Faradji, Albert; Peynet, Jocelyne; Costagliola, Dominique

2002-02-01

Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma-derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin-stabilized recombinant factor and plasma-derived factor, we studied the prevalence of IgG antibodies to B19 (anti-B19) in 193 haemophiliac children between 1 and 6-years of age who had previously been treated with albumin-stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti-B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti-B19 was found in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates than in children treated with albumin- stabilized recombinant FVIII only (OR: 22.3; CI: 7.9-62.8), independently of the other factors studied.

Immunomodulatory effects of OX40Ig gene-modified adipose tissue-derived mesenchymal stem cells on rat kidney transplantation

OpenAIRE

Liu, Tao; Zhang, Yue; Shen, Zhongyang; Zou, Xunfeng; Chen, Xiaobo; Chen, Li; Wang, Yuliang

2016-01-01

Recent studies have suggested that adipose tissue-derived mesenchymal stem cell (ADSC) therapy and OX40 costimulation blockade are two immunomodulatory strategies used to suppress the immune response to alloantigens. However, relatively little has been reported regarding the immunomodulatory potential of the abilityof these two strategies to synergize. Thus, in the present study, we aimed to investigate OX40-Ig fusion protein (OX40Ig) expression in ADSCs and to validate their more potent immu...

Derivation of the Radioactivity Index for Consumer Goods Containing NORM

Energy Technology Data Exchange (ETDEWEB)

Jang, Mee; Chung, Kun Ho; Ji, Young Yong; Kim, Chang Jong; Kang, Mun Ja [KAERI, Daejeon (Korea, Republic of)

2016-05-15

Some consumer goods to promote health such as anion bracelets, necklace and mats contain naturally occurring radioactive material (NORM). Some of them can cause problems because of high radioactivity. In the regulations, there is an annual effective dose limit of 1mSv for products, but the activity concentration limits and radioactivity index for products is not established yet. Although there are few researches for consumer goods containing NORM in foreign countries, in Japan, for the consumer goods. To regulate the NORM in consumer goods, it is necessary to derive activity concentration limits corresponding to the annual limits of 1mSv. In this research, we calculated the activity concentration limits according to the usage quantities of consumer goods. Using these results, it is possible to suggest several radioactivity indexes to apply to a lot of consumer goods.

Derivation of the Radioactivity Index for Consumer Goods Containing NORM

International Nuclear Information System (INIS)

Jang, Mee; Chung, Kun Ho; Ji, Young Yong; Kim, Chang Jong; Kang, Mun Ja

2016-01-01

Some consumer goods to promote health such as anion bracelets, necklace and mats contain naturally occurring radioactive material (NORM). Some of them can cause problems because of high radioactivity. In the regulations, there is an annual effective dose limit of 1mSv for products, but the activity concentration limits and radioactivity index for products is not established yet. Although there are few researches for consumer goods containing NORM in foreign countries, in Japan, for the consumer goods. To regulate the NORM in consumer goods, it is necessary to derive activity concentration limits corresponding to the annual limits of 1mSv. In this research, we calculated the activity concentration limits according to the usage quantities of consumer goods. Using these results, it is possible to suggest several radioactivity indexes to apply to a lot of consumer goods

Genetic Recombination

Science.gov (United States)

Whitehouse, H. L. K.

1973-01-01

Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers

International Nuclear Information System (INIS)

Roth, M.; Emmons, L.R.; Perruchoud, A.; Block, L.H.

1991-01-01

The plausible role that platelet-derived growth factor (PDGF) has in the localized pathophysiological changes that occur in the arterial wall during development of atherosclerotic lesions led the authors to investigate the influence of recombinant (r)PDGF isomers -AA, -AB, and -BB on the expression of low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG0CoA) reductase [(S)-mevalonate:NAD + oxidoreductase (CoA-acylating), EC 1.1.1.88] genes. In addition, they clarified the role of protein kinase C (PKC) in expression of the two genes in human skin fibroblasts and vascular smooth muscle cells. The various rPDGF isoforms are distinct in their ability to activate transcription of both genes: (i) both rPDGF-AA and -BB stimulate transcription of the LDL-R gene; in contrast, rPDGF-BB but not -AA, activates transcription of the HMG-CoA reductase gene; (ii) all recombinant isoforms of PDGF activate transcription of the c-fos gene; (iii) while rPDGF-dependent transcription of the lDL-R gene occurs independently of PKC, transcription of the HMG-CoA reductase gene appears to involve the action of that enzyme

Enhancement of SV40 transformation by treatment of C3H2K cells with uv light and caffeine. I. Combined effect of uv light and caffeine

International Nuclear Information System (INIS)

Ide, T.; Anzai, K.; Andoh, T.

1975-01-01

Treatment of cultured mouse cells, C3H2K, with uv light and/or caffeine enhanced the frequency of SV40-induced transformation. This enhancement depends upon the doses of uv and caffeine and the mode of combination of these agents. Irradiation of cells with increasing doses of uv just before infection resulted in approximately 2-fold enhancement of the transformation frequency up to a dose of 90 ergs/mm 2 and 3.3-fold at 150 ergs/mm 2 . Addition of 1 mM caffeine to the medium for 4 days subsequent to infection brought about a 2-fold enhancement. When cells were irradiated and treated with 1 mM caffeine, the enhancement was approximately 4-fold up to a uv dose of 90 ergs/mm 2 and 5.9-fold at 150 ergs/mm 2 . When 0.1 to 4 mM caffeine was added for 4 days postinfection, the absolute number of transformations increased, and an enhancement ratio of 1.3 to 6.8 resulted. After the addition of the same increasing doses of caffeine to uv-irradiated cells (75 ergs/mm 2 ), the enhancement of transformation frequency was even higher ranging 2.0 to 13.3. The transformation frequencies thus obtained by the double treatment were always higher than those predicted if uv and caffeine acted additively. The transformation frequency was little affected by the addition of dibutyrylcyclic AMP and theophylline

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Directory of Open Access Journals (Sweden)

M.C.S. Brum

2010-02-01

Full Text Available Bovine herpesvirus type 5 (BoHV-5 is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE or thymidine kinase (TK gene or both (gE/TK from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99. A gE-deleted recombinant virus (BoHV-5 gE∆ was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆ was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE BoHV-5 recombinant (BoHV-5 gE/TK∆ was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK cells, the mutants lacking gE (BoHV-5 gE∆ and TK + gE (BoHV-5 gE/TK∆ produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆ were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆ produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Uranium, radium and 40K isotopes in bottled mineral waters from Outer Carpathians, Poland

International Nuclear Information System (INIS)

Kozlowska, B.; Walencik, A.; Dorda, J.; Przylibski, T.A.

2007-01-01

Radioactivity content in commercially bottled mineral waters from Outer Carpathians was investigated on the basis of 28 samples. Activity concentration results for radium isotopes 226,228 Ra, uranium isotopes 234,238 U and isotopic ratios 234 U/ 238 U were determined. The correlations between investigated isotopes and calculated potassium 40 K ions dissolved in water were carried out. The results show a correlation between TDS (total dissolved solids) values and dissolved radionuclides. High correlation coefficients were observed between total radium content and 40 K. The isotopic ratio of 234 U/ 238 U varies in the range from 1.6 to 7 in all investigated waters which means that there is no radioactive equilibrium between the parent nuclide 238 U and its daughter 234 U. The effective radiation dose coming from studied radium and uranium radionuclides consumed with mineral water from the Outer Carpathians obtained by a statistical Pole is equal to 4.3μSv/year (58 l/year water consumption) and do not exceed the permissible limit equal to 100μSv/year. Assuming 0.5 l consumption per day, i.e. 182.5 l/year, the effective dose is equal to 13.4μSv/year, what is still below the unit

Evaluation of wheat stillage for ethanol production by recombinant Zymomonas mobilis

Energy Technology Data Exchange (ETDEWEB)

Davis, L.; Peiris, P. [University of Western Sydney, Penrith (Australia). School of Science, Food and Horticulture; Young-Jae Jeon; Svenson, C.; Rogers, P. [University of New South Wales, Sydney (Australia). School of Biotechnology and Biomolecular Sciences; Pearce, J. [Manildra Group, Bomaderry (Australia)

2005-07-01

Stillage is the main residue from the starch-to-ethanol fermentation process.Carbohydrates (hemicellulose and cellulose) comprise approximately 50% (w/w)of the total components of stillage. Conversion of the hemicellulose and cellulose to fermentable sugars and then to ethanol has the potential to significantly increase the efficiency of the process. The hydrolysis of stillage to fermentable sugars was optimised using 2% (v/v) H{sub 2}SO{sub 4} at 100{sup o}C for 5.5 h and produced 18 g/L xylose, 11.5 g/L arabinose and 6.5 g/L glucose from 120 g/L stillage. Further hydrolysis using enzymes increased the release of glucose by 61%. Furfural, acetate and lactate were the main inhibitors present in the acid hydrolysate of stillage. The lignin-derived inhibitors hydroxymethylfuraldehyde, hydroxybenzaldehyde, vanillin and syringaldehyde were not detected. Neutralisation of the hydrolysate with lime to pH 5 decreased the concentration of furfural by 50%. Fermentation of hydrolysate supplemented with glucose 10 g/L, by recombinant Zymomonas mobilis ZM4(pZB5), produced 11 g/L of ethanol after 70 h, with residual xylose 12 g/L. Supplementation of the hydrolysate with 5 g/L yeast extract and 40 g/L glucose produced 28 g/L ethanol with 2.6 g/L residual xylose after 18 h. Arabinose was not utilised by this particular recombinant strain. From the results, Z. mobilis ZM4(pZB5) may be a suitable candidate for the fermentation of both glucose and xylose in stillage acid hydrolysates. (author)

Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China

Science.gov (United States)

Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.

2000-01-01

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

Distribution of ultraviolet-induced lesions in Simian Virus 40 DNA

International Nuclear Information System (INIS)

Bourre, F.; Renault, G.; Sarasin, A.; Seawell, P.C.

1985-01-01

In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000J/m 2 , our results show that the formation frequency of thymine-thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight in the molecular mechanisms of UV-mutagenesis

The adaptation of limb kinematics to increasing walking speeds in freely moving mice 129/Sv and C57BL/6.

Science.gov (United States)

Serradj, Nadjet; Jamon, Marc

2009-07-19

The kinematics of locomotion was analyzed in two strains of great importance for the creation of mutated mice (C56BL/6 and 129/Sv). Different behavioral situations were used to trigger sequences of movement covering the whole range of velocities in the mice, and the variations of kinematic parameters were analyzed in relation with velocity. Both stride frequency and stride length contributed to the moving speed, but stride frequency was found to be the main contributor to the speed increase. A trot-gallop transition was detected at speed about 70 cm/s, in relation with a sharp shift in limb coordination. The results of this study were consistent with pieces of information previously published concerning the gait analyses of other strains, and provided an integrative view of the basic motor pattern of mice. On the other hand some qualitative differences were found in the movement characteristics of the two strains. The stride frequency showed a higher contribution to speed in 129/Sv than in C57BL/6. In addition, 129/Sv showed a phase shift in the forelimb and hindlimb, and a different position of the foot during the stance time that revealed a different gait and body position during walking. Overall, 129/Sv moved at a slower speed than C57BL/6 in any behavioral situation. This difference was related to a basal lower level of motor activity. The possibility that an alteration in the dopamine circuit was responsible for the different movement pattern in 129/Sv is discussed.

Cat odor exposure induces distinct changes in the exploratory behavior and Wfs1 gene expression in C57Bl/6 and 129Sv mice.

Science.gov (United States)

Raud, Sirli; Sütt, Silva; Plaas, Mario; Luuk, Hendrik; Innos, Jürgen; Philips, Mari-Anne; Kõks, Sulev; Vasar, Eero

2007-10-16

129Sv and C57Bl/6 (Bl6) strains are two most widely used inbred mice strains for generation of transgenic animals. The present study confirms the existence of substantial differences in the behavior of these two mice strains. The exploratory behavior of Bl6 mice in a novel environment was significantly higher compared to 129Sv mice. The exposure of mice to cat odor-induced an anxiety-like state in Bl6, but not in 129Sv mice. The levels of Wfs1 gene expression did not differ in the prefrontal cortex, mesolimbic area and temporal lobe of experimentally naive Bl6 and 129Sv mice. However, after cat odor exposure the expression of Wfs1 gene was significantly lower in the mesolimbic area and temporal lobe of Bl6 mice compared to 129Sv strain. Dynamics of Wfs1 gene expression and exploratory behavior suggest that the down-regulation of Wfs1 gene in Bl6 mice might be related to the increased anxiety. Further studies are needed to test the robustness and possible causal relationship of this finding.

Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of Wagyu compared with those of Angus cattle.

Science.gov (United States)

Wei, S; Fu, X; Liang, X; Zhu, M J; Jiang, Z; Parish, S M; Dodson, M V; Zan, L; Du, M

2015-03-01

cells was lower than that of Angus, providing potential clues for breed differences on proliferation of SV cells in these two cattle breeds. The results of this study suggest that subcutaneous adipose-derived SV cells of Wagyu possess a lower trend of adipogenesis but higher mitogenesis compared with those of Angus.

A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system.

Science.gov (United States)

Tang, Na; Zhang, Yaoyao; Pedrera, Miriam; Chang, Pengxiang; Baigent, Susan; Moffat, Katy; Shen, Zhiqiang; Nair, Venugopal; Yao, Yongxiu

2018-01-29

Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines. Copyright © 2018 The Pirbright Institute. Published by Elsevier Ltd.. All rights reserved.

210Po, 210Pb, 40K and 137Cs in edible wild berries and mushrooms and ingestion doses to man from high consumption rates of these wild foods

International Nuclear Information System (INIS)

Gwynn, Justin P.; Nalbandyan, Anna; Rudolfsen, Geir

2013-01-01

This paper discusses activity concentrations of 210 Po, 210 Pb, 40 K and 137 Cs in edible wild berries and mushrooms collected from Øvre Dividalen national park, Northern Norway and derives committed effective ingestion doses to man based on high consumption rates of these wild foods. Edible wild berries and mushrooms accumulated similar levels of 210 Pb, but mushrooms accumulated higher levels of 210 Po and 40 K than berries. There appears to be a clear difference in the ability of Leccinum spp. of fungi to accumulate 210 Po and/or translocate 210 Po to mushrooms compared to Russula spp. of fungi. Activity concentrations of 137 Cs in edible wild berries and mushrooms from Øvre Dividalen national park reflected the lower levels of fallout of this radionuclide in Northern Norway compared to more central areas following the Chernobyl accident. For mushrooms, ingestion doses are dominated by 210 Po, while for berries, 40 K is typically the main contributor to dose. Based on high consumption rates, ingestion doses arising from the combination of 210 Po, 210 Pb and 40 K were up to 0.05 mSv/a for berries and 0.50 mSv/a for mushrooms. Consumption of such wild foods may result in a significant contribution to total annual doses when consumed in large quantities, particularly when selecting mushrooms species that accumulate high activity concentrations of 210 Po. - Highlights: ► 210 Po/ 210 Pb activity ratios were typically less than one for berries. ► 210 Po/ 210 Pb activity ratios were all greater than one for mushrooms. ► Dose rates from mushrooms were dominated by 210 Po and by 40 K for berries. ► Wild foods can give a significant contribution to total annual ingestion dose.

Quantum electrodynamic theory of recombination of an electron with a highly charged ion

International Nuclear Information System (INIS)

Shabaev, V.M.

1994-01-01

The consequent quantum electrodynamic theory of the process of the recombination of an electron with a multicharged ion is considered. The reduction technique for the calculation of this process by perturbation theory is formulated. The process of the recombination of an electron with a very highly charged one-electron ion for the case of resonance with the doubly excited (2s,2s) 0 , (2p 1/2 ,2p 1/2 ) 0 , (2s,2p 1/2 ) 0,1 states is studied. The formulas for the cross section of the process are derived for two possible versions of the experiment. The interference between the radiative-recombination process and the dielectronic-recombination (DR) process, and the interference between the DR amplitudes for the levels with the identical quantum numbers [(2s,2s) 0 , (2p 1/2 ) 0 ] are taken into account. The deviation of the shape of the resonances from the Lorentz one, due to the interference terms, is discussed

SV3R : un framework pour la gestion de la variabilité des services

Directory of Open Access Journals (Sweden)

Boutaina Chakir

2014-11-01

Full Text Available The emergence and expansion of the development paradigm based on service-oriented approaches have been behind the elaboration of new models and methods that aim at facilitating the reuse of services within multiples context of use. This requires providing systematically services with several possible realizations. Among the promising approaches that achieve this objective is the management of variability which has been widely adopted by the software engineering disciplines and whose objective is to facilitate the adaptation or the configuration of software artifacts in a systematic way. Hence, in this work we provide a framework for the development (for and by reuse of services supporting variability, called “SV3R” (Service Variability Representation and Resolution for Reuse. This paper introduces at first the concept of variability. Afterwards, it presents some related work, before giving an overview of the framework SV3R and describing

Radio recombination lines from H II regions

International Nuclear Information System (INIS)

Silverglate, P.R.

1978-01-01

Radio recombination lines have been observed from forty-six H II regions. The Arecibo 1000-foot radio telescope was used to provide high sensitivity and high angular resolution at 1400 MHz (gain approx. 7.7 0 K/Jy, HPBW = 3:2) and 2372 MHZ (gain approx. 6.3 0 K/Jy, HPBW = 2'). Observations were made at 1400 MHz in the frequency switching mode, and at 2372 MHz in the total power mode. Gaussians were fit to be observed lines to derive velocities, line widths, and line temperatures. From the velocities kinematic distances were derived. For eleven sources H I absorption measurements were also made. The absorption spectra enabled the kinematic distance ambiguity to be resolved for some sources. The absorption spectra themselves were found to have extremely sharp, non-gaussian edges. One explanation for these is a model where the interstellar medium contains many H I cloudlets with T/sub s/less than or equal to 100 0 K and turbulent velocities less than or equal to 3 km/s. The H I absorption spectrum is then a superposition of many narrow gaussian profiles. It was also found from a comparison of H I absorption velocities with radio recombination line velocities that peculiar motions exist in the interstellar medium with velocities of up to 10 km/s. Using the measured line temperatures and continuum temperatures, estimates were desired of emission measures, electron temperatures, and electron densities, using a non-LTE analysis. Non-LTE effects were important only for the hottest and densest H II regions. The non-LTE calculations were checked through a comparison derivation of electron temperatures using hydrogen beta lines

Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

Science.gov (United States)

Almeida, Aline G; Pinto, Rodrigo C V; Smales, C Mark; Castilho, Leda R

2017-08-01

To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8 °C. NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance. Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

Reflection of P and SV waves at the free surface of a monoclinic ...

Indian Academy of Sciences (India)

The propagation of plane waves in an anisotropic elastic medium possessing monoclinic symmetry is discussed. The expressions for the phase velocity of qP and qSV waves propagating in the plane of elastic symmetry are obtained in terms of the direction cosines of the propagation vector. It is shown that, in general, ...

Orwellův svět v roce 2009

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Miloslav Petrusek

2017-10-01

Full Text Available V červnu 1949 vyšla v nakladatelství Harcourt, Brace v New Yorku kniha, která se zařadila mezi několik zásadních beletristických děl 20. století. Svým politickým vyzněním měla otřást totalitními režimy, tímto zrůdným plodem „instrumentální racionality“ a ad absurdum dovedené byrokracie. Pomineme literaturu, jejímž dominantním literárním tématem je kritika nacismu – třebas Feuchtwangerovu triologii, román I ve smrti sami, Mannův esej Bratr Hitler, ale i alegorický příběh Doktora Fausta či nejslavnější román Heinricha Manna Mefisto, případně kritika latinskoamerických autoritářských režimů – třebas vynikající román Marquézův Podzim patriarchy nebo romány Carpentierovy Výbuch v katedrále a Náprava dle metody. Setrváme především u literárního obrazu a kritiky sovětského systému. Je jistě věcí individuální zkušenosti (osobní i literární a do značné míry subjektivní volby, která díla vybereme. Zdá se nicméně, že nejméně tři patří k neopominutelné „klasice“. Jsou totiž nejen formálně často jmenována, ale stále čtena, jsou živá a zřejmě tedy obsahují jakýsi aktualizující náboj, který jim nedovolí zůstat v řadě strnulé nehybnosti nečtených, leč ctěných klasiků. Myslím, že jde o knihu Artura Koestlera Tma o polednách, která již v roce 1939 odhalila – bez znalosti jakýchkoliv svědeckých či archivních podkladů – mechanismus moskevských procesů, které „otřásly světem“, o žánrově science fiction s titulem 1984 George Orwella a konečně o Solženicynův monument Soustroví Gulag.

Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

Science.gov (United States)

Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

2015-04-10

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

Science.gov (United States)

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-03-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

Directory of Open Access Journals (Sweden)

Wendy Gibson

2015-03-01

Full Text Available Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr responsible for sleeping sickness (Human African Trypanosomiasis, HAT in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Freeman, Kathryn M. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States); Hoffmann, George R. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States)]. E-mail: ghoffmann@holycross.edu

2007-03-01

Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, {beta}-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv{sup +} revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state.

Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Freeman, Kathryn M.; Hoffmann, George R.

2007-01-01

Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, β-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv + revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state

Yeast-recombinant hepatitis B vaccine: efficacy with hepatitis B immune globulin in prevention of perinatal hepatitis B virus transmission

International Nuclear Information System (INIS)

Stevens, C.E.; Taylor, P.E.; Tong, M.J.; Toy, P.T.; Vyas, G.N.; Nair, P.V.; Weissman, J.Y.; Krugman, S.

1987-01-01

A yeast-recombinant hepatitis B vaccine was licensed recently by the Food and Drug administration and is now available. To assess the efficacy of the yeast-recombinant vaccine, the authors administered the vaccine in combination with hepatitis B immune globulin to high-risk newborns. If infants whose mothers were positive for both hepatitis B surface antigen and the e antigen receive no immunoprophylaxis, 70% to 90% become infected with the virus, and almost all become chronic carriers. Among infants in this study who received hepatitis B immune globulin at birth and three 5- + g doses of yeast-recombinant hepatitis B vaccine, only 4.8% became chronic carriers, a better than 90% level of protection and a rate that is comparable with that seen with immune globulin and plasma-derived hepatitis B vaccine. Hepatitis surface antigen and antibodies were detected by radioimmunoassay. These data suggest that, in this high-risk setting, the yeast-recombinant vaccine is as effective as the plasma-derived vaccine in preventing hepatitis B virus infection and the chronic carrier state

Registration of Wyandot × PI 567301B soybean recombinant inbred line population

Science.gov (United States)

A soybean [Glycine max (L.) Merr] mapping population (Reg. No., SNL MAP) consisting of 357 F7-derived recombinant inbred lines (RILs) was jointly developed by the USDA-Agricultural Research Service and the Ohio Agricultural Research and Development Center (OARDC) in Wooster, OH. The population was ...

Malignant transformation of guinea pig cells after exposure to ultraviolet-irradiated guinea pig cytomegalovirus

International Nuclear Information System (INIS)

Isom, H.C.; Mummaw, J.; Kreider, J.W.

1983-01-01

Guinea pig cells were malignantly transformed in vitro by ultraviolet (uv)-irradiated guinea pig cytomegalovirus (GPCMV). When guinea pig hepatocyte monolayers were infected with uv-irradiated GPCMV, three continuous epithelioid cell lines which grew in soft agarose were established. Two independently derived GPCMV-transformed liver cells and a cell line derived from a soft agarose clone of one of these lines induced invasive tumors when inoculated subcutaneously or intraperitoneally into nude mice. The tumors were sarcomas possibly derived from hepatic stroma or sinusoid. Transformed cell lines were also established after infection of guinea pig hepatocyte monolayers with human cytomegalovirus (HCMV) or simian virus 40 (SV40). These cell lines also formed colonies in soft agarose and induced sarcomas in nude mice. It is concluded that (i) GPCMV can malignantly transform guinea pig cells; (ii) cloning of GPCMV-transformed cells in soft agarose produced cells that induced tumors with a shorter latency period but with no alteration in growth rate or final tumor size; and (iii) the tumors produced by GPCMV-and HCMV-transformed guinea pig cells were more similar to each other in growth rate than to those induced by SV40-transformed guinea pig cells

Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

Science.gov (United States)

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line

2014-01-01

ABSTRACT Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. PMID:25096874

Recombinant proteins of helminths with immunoregulatory properties and their possible therapeutic use.

Science.gov (United States)

Nascimento Santos, Leonardo; Carvalho Pacheco, Luis Gustavo; Silva Pinheiro, Carina; Alcantara-Neves, Neuza Maria

2017-02-01

The inverse relationship between helminth infections and the development of immune-mediated diseases is a cornerstone of the hygiene hypothesis and studies were carried out to elucidate the mechanisms by which helminth-derived molecules can suppress immunological disorders. These studies have fostered the idea that parasitic worms may be used as a promising therapeutic alternative for prevention and treatment of immune-mediated diseases. We discuss the current approaches for identification of helminth proteins with potential immunoregulatory properties, including the strategies based on high-throughput technologies. We also explore the methodological approaches and expression systems used for production of the recombinant forms of more than 20 helminth immunomodulatory proteins, besides their performances when evaluated as immunotherapeutic molecules to treat different immune-mediated conditions, including asthma and inflammatory bowel diseases. Finally, we discuss the perspectives of using these parasite-derived recombinant molecules as tools for future immunotherapy and immunoprophylaxis of human inflammatory diseases. Copyright © 2016. Published by Elsevier B.V.

Photoionization and electron-ion recombination of Fe XVII for high temperature plasmas

International Nuclear Information System (INIS)

Nahar, Sultana N.

2012-01-01

Earlier studies on electron-ion recombination of Fe XVII, e+FeXVIII→FeXVII, concentrated on low temperature region. However, due to its higher abundance, recombination in the high temperature region is of great importance. Total and level-specific recombination cross sections and rates of Fe XVII are presented from the detailed study in the high temperature. The calculations were carried out using the unified method which incorporates both the radiative recombination (RR) and dielectronic recombination (DR) including the interference effects. The method also yields self-consistent set of recombination rates and photoionization cross sections. Unified method is implemented through relativistic Breit-Pauli R-matrix (BPRM) method and close coupling (CC) approximation. For the details of the high energy and high temperature features a CC wave function expansion consisting of 60 levels from n=2 and 3 complexes of the core Fe XVIII was considered. Earlier study included core excitations to n=2 levels only. It is found that the resonances due to core excitations to n=3 levels are much more extensive and stronger than those to n=2 levels and increase the recombination considerably in the high temperature region. While earlier study of 3-level calculations agree very well with the experimentally derived low temperature recombination, the high temperature rate shows a broad peak at about 5×10 6 K, near the maximum abundance of the ion, due to dominance of DR via PEC (photo-excitation-of-core) resonances of n=3 levels. Level-specific recombination rate coefficients, which include both the RR and DR, are presented for 454 levels (n≤10, l≤9, 0 ≤J≤8 with even and odd parities) of Fe XVII. This is the first large-scale BPRM calculations for recombination of a complex atomic system beyond He- and Li-like ions. The results are expected to be accurate with 10-20% uncertainty and provide accurate modelings of ultraviolet to X-ray spectra.

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

Science.gov (United States)

Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

2015-01-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

Laser-induced electron--ion recombination used to study enhanced spontaneous recombination during electron cooling

International Nuclear Information System (INIS)

Schramm, U.; Wolf, A.; Schuess ler, T.; Habs, D.; Schwalm, D.; Uwira, O.; Linkemann, J.; Mueller, A.

1997-01-01

Spontaneous recombination of highly charged ions with free electrons in merged velocity matched electron and ion beams has been observed in earlier experiments to occur at rates significantly higher than predicted by theoretical estimates. To study this enhanced spontaneous recombination, laser induced recombination spectra were measured both in velocity matched beams and in beams with well defined relative velocities, corresponding to relative electron-ion detuning energies ranging from 1 meV up to 6.5 meV where the spontaneous recombination enhancement was found to be strongly reduced. Based on a comparison with simplified calculations, the development of the recombination spectra for decreasing detuning energies indicates additional contributions at matched velocities which could be related to the energy distribution of electrons causing the spontaneous recombination rate enhancement

Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

International Nuclear Information System (INIS)

Fraenkel, A.H.M.

1974-01-01

Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

Science.gov (United States)

Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

2016-02-01

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. © FASEB.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

Science.gov (United States)

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

Molecular and Recombinational Mapping of Mutations in the Ace Locus of Drosophila melanogaster

OpenAIRE

Nagoshi, Rodney N.; Gelbart, William M.

1987-01-01

The Ace locus in Drosophila melanogaster is known to be the structural gene for acetylcholinesterase. Ace is located in a region of chromosome arm 3R which has been subjected to intensive genetic and molecular analysis. Previous deletion mapping studies have identified a 40-kb region within which the Ace gene resides. This report focuses on the further localization of Ace within this 40-kb interval. Within this region, selective fine structure recombinational analysis was employed to localize...

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzenemethanaminium, N-(3-aminopropyl...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzenemethanaminium, N-(3-aminopropyl)-N,N...

Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis

Directory of Open Access Journals (Sweden)

Frøkiær Hanne

2007-08-01

Full Text Available Abstract Background Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion Recombinant production of Ara h 2 using L. lactis can offer high yields

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

Energy Technology Data Exchange (ETDEWEB)

Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

2015-07-31

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

International Nuclear Information System (INIS)

Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka

2015-01-01

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS SV40 ) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS SV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS SV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS SV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS SV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

Science.gov (United States)

Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

2015-10-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Killing Effect of Ad5/F35-APE1 siRNA Recombinant Adenovirus in Combination with Hematoporphrphyrin Derivative-Mediated Photodynamic Therapy on Human Nonsmall Cell Lung Cancer

Directory of Open Access Journals (Sweden)

Lei Xia

2013-01-01

Full Text Available The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group ( after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (. The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI. In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

Jeans instability of inhomogeneous dusty plasma with polarization force, ionization and recombination

International Nuclear Information System (INIS)

Jain, Shweta; Sharma, Prerana; Chhajlani, R K

2017-01-01

The self-gravitational Jeans instability has been studied in dusty plasma containing significant background of neutral pressure and recombination of ions and electrons on the dust surface. The full dynamics of charged dust grains, ions and neutral species are employed considering the electrons as Maxwellian. We have derived the general dispersion relation for collisional dusty plasma with ionization, recombination and polarization force. The general dispersion relation describes the effects of considered parameters which are solved in different dusty plasma situations. Further, the dispersion relation is solved numerically. The present work is applicable to understand the structure formation of interstellar molecular clouds in astrophysical plasma. (paper)

Jeans instability of inhomogeneous dusty plasma with polarization force, ionization and recombination

Science.gov (United States)

Jain, Shweta; Sharma, Prerana; Chhajlani, R. K.

2017-05-01

The self-gravitational Jeans instability has been studied in dusty plasma containing significant background of neutral pressure and recombination of ions and electrons on the dust surface. The full dynamics of charged dust grains, ions and neutral species are employed considering the electrons as Maxwellian. We have derived the general dispersion relation for collisional dusty plasma with ionization, recombination and polarization force. The general dispersion relation describes the effects of considered parameters which are solved in different dusty plasma situations. Further, the dispersion relation is solved numerically. The present work is applicable to understand the structure formation of interstellar molecular clouds in astrophysical plasma.

Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

International Nuclear Information System (INIS)

Perez, C.F.

1984-08-01

The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT + colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references

Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

Energy Technology Data Exchange (ETDEWEB)

Perez, C.F.

1984-08-01

The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments

NARCIS (Netherlands)

F. Aladin (Farah); A.W.C. Einerhand (Sandra); J. Bouma (Janneke); S. Bezemer (Sandra); P. Hermans (Pim); D. Wolvers (Danielle); K. Bellamy (Kate); L.G.J. Frenken (Leon); J. Gray (Jim); M. Iturriza-Gómara (Miren)

2012-01-01

textabstractRotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment

Nanobodies and recombinant binders in cell biology.

Science.gov (United States)

Helma, Jonas; Cardoso, M Cristina; Muyldermans, Serge; Leonhardt, Heinrich

2015-06-08

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.

Nanobodies and recombinant binders in cell biology

Science.gov (United States)

Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

2015-01-01

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

Validation of the portuguese version of the tampa scale for kinesiophobia heart (TSK-SV heart

Directory of Open Access Journals (Sweden)

Gabriela Lima de Melo Ghisi

Full Text Available ABSTRACT Introduction: It has been shown that kinesiophobia has a negative influence on the outcomes of cardiac rehabilitation and consequently is important for the clinical setting. Objective: The objective of this study was to translate, culturally adapt, and psychometrically validate the Tampa Scale for Kinesiophobia Heart (TSK-SV Heart to Brazilian Portuguese. Methods: The Portuguese version was tested in 300 patients in cardiac rehabilitation. Test-retest reliability was assessed by intraclass correlation coefficient, internal consistency by Cronbach’s alpha, and criterion validity was assessed with respect to patients’ education, income, duration of cardiac rehabilitation, and sex. Results: After intraclass correlation coefficient analysis, one item was excluded. All four areas were considered internally consistent (α >0.7. Significant differences between mean total scores and income (p 37. Conclusions: The Brazilian Portuguese version of TSK-SV Heart demonstrated sufficient reliability, consistency and validity, supporting its use in future studies.

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

Science.gov (United States)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Recombination homeostasis of meiosis during spermatogenesis under nicotine treatment

Directory of Open Access Journals (Sweden)

Zhai Jingli

2018-01-01

Full Text Available Cigarette smoking can affect male fertility via the quality of semen. To explore the effects of nicotine, a major component of cigarettes, on meiotic recombination during spermatogenesis, C57BL/6J male mice were injected with nicotine at a dosage of 0.2 mg/100 g body weight daily for 35 days (nicotine-treated group; mice in the control group were injected with isopycnic normal saline. According to previous expression profiles of mouse sperm, a subset of meiosis-related genes was pooled using bioinformatic analysis. Protein expression was compared between the two groups using by Western blotting and immunohistochemistry. Recombination frequency during the meiosis phase of spermatogenesis was estimated by combined use of chromosome spread and immunofluorescence staining in mouse testes. Data mining analysis indicated that 4 genes that express meiotic topoisomerase-like protein SPO11, MutS protein homolog 4 (MSH4, strand exchange protein RAD51 and MutL protein homologue 1 (MLH1, were associated with the meiosis recombination process. The results of Western blotting and immunohistochemistry further showed that the protein expression of SPO11 (0.73-fold and MSH4 (0.73-fold was downregulated in murine testes after nicotine treatment, whereas the protein expression of both RAD51 (2.06-fold and MLH1 (1.40-fold was upregulated. Unexpectedly, we did not detect a significant difference in recombination frequency in meiosis during spermatogenesis in the nicotine-treated group as compared to the control. Taken together, these results indicate that nicotine can affect the expression profile of restructuring-related genes, but it does not significantly change the recombination frequency during male meiosis. These findings suggest there is a self-regulating mechanism during meiotic chromosome restructuring in male mice that responds to environmental stress.

Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies.

Science.gov (United States)

Lee, Warren; Syed Atif, Ali; Tan, Soo Choon; Leow, Chiuan Herng

2017-08-01

The advantages of chicken (Gallus gallus domesticus) antibodies as immunodiagnostic and immunotherapeutic biomolecules has only been recently recognized. Even so, chicken antibodies remain less-well characterized than their mammalian counterparts. This review aims at providing a current overview of the structure, function, development and generation of chicken antibodies. Additionally, brief but comprehensive insights into current knowledge pertaining to the immunogenetic framework and diversity-generation of the chicken immunoglobulin repertoire which have contributed to the establishment of recombinant chicken mAb-generating methods are discussed. Focus is provided on the current methods used to generate antibodies from chickens with added emphasis on the generation of recombinant chicken mAbs and its derivative formats. The advantages and limitations of established protocols for the generation of chicken mAbs are highlighted. The various applications of recombinant chicken mAbs and its derivative formats in immunodiagnostics and immunotherapy are further detailed. Copyright © 2017 Elsevier B.V. All rights reserved.

Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

DEFF Research Database (Denmark)

Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

1996-01-01

substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types...... of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV...

SvO(2)-guided resuscitation for experimental septic shock: effects of fluid infusion and dobutamine on hemodynamics, inflammatory response, and cardiovascular oxidative stress.

Science.gov (United States)

Rosário, André Loureiro; Park, Marcelo; Brunialti, Milena Karina; Mendes, Marialice; Rapozo, Marjorie; Fernandes, Denise; Salomão, Reinaldo; Laurindo, Francisco Rafael; Schettino, Guilherme Paula; Azevedo, Luciano Cesar P

2011-12-01

The pathogenetic mechanisms associated to the beneficial effects of mixed venous oxygen saturation (SvO(2))-guided resuscitation during sepsis are unclear. Our purpose was to evaluate the effects of an algorithm of SvO(2)-driven resuscitation including fluids, norepinephrine and dobutamine on hemodynamics, inflammatory response, and cardiovascular oxidative stress during a clinically resembling experimental model of septic shock. Eighteen anesthetized and catheterized pigs (35-45 kg) were submitted to peritonitis by fecal inoculation (0.75 g/kg). After hypotension, antibiotics were administered, and the animals were randomized to two groups: control (n = 9), with hemodynamic support aiming central venous pressure 8 to 12 mmHg, urinary output 0.5 mL/kg per hour, and mean arterial pressure greater than 65 mmHg; and SvO(2) (n = 9), with the goals above, plus SvO(2) greater than 65%. The interventions lasted 12 h, and lactated Ringer's and norepinephrine (both groups) and dobutamine (SvO(2) group) were administered. Inflammatory response was evaluated by plasma concentration of cytokines, neutrophil CD14 expression, oxidant generation, and apoptosis. Oxidative stress was evaluated by plasma and myocardial nitrate concentrations, myocardial and vascular NADP(H) oxidase activity, myocardial glutathione content, and nitrotyrosine expression. Mixed venous oxygen saturation-driven resuscitation was associated with improved systolic index, oxygen delivery, and diuresis. Sepsis induced in both groups a significant increase on IL-6 concentrations and plasma nitrate concentrations and a persistent decrease in neutrophil CD14 expression. Apoptosis rate and neutrophil oxidant generation were not different between groups. Treatment strategies did not significantly modify oxidative stress parameters. Thus, an approach aiming SvO(2) during sepsis improves hemodynamics, without any significant effect on inflammatory response and oxidative stress. The beneficial effects associated

Performance characterization of hydrogen isotope exchange and recombination catalysts for tritium processing

International Nuclear Information System (INIS)

Suppiah, S.; Ryland, D.; Marcinkowska, K.; Boniface, H.; Everatt, A.

2010-01-01

AECL's hydrogen isotope exchange catalyst and recombination catalysts have been successfully applied to a wide range of industrial tritium-removal applications. The catalysts are used for Liquid Phase Catalytic Exchange (LPCE) and for gas-phase and trickle-bed recombination of hydrogen isotopes and have led to process simplification, improved safety and operational advantages. Catalyst performance design equations derived from laboratory testing of these catalysts have been validated against performance under industrial conditions. In a Combined Electrolysis and Catalytic Exchange (CECE) demonstration plant analyses of LPCE and recombiner efficiency were carried out as a function of catalyst activity over a wide range of operation. A steady-state process simulation used to model and design the hydrogen-water isotopic exchange processes, such as the CECE detritiation plant, was validated using the results of this demonstration. Catalyst development for isotope-exchange and recombination applications has continued over the last decade. As a result, significant improvements in catalyst performance have been achieved for these applications. This paper outlines the uniqueness of AECL's specialized catalysts and process designs for these applications with examples from laboratory and industrial case studies.

[Study on the movement of the carrier recombination region in organic light-emitting diodes (OLEDs) based on DPVBi/Alq3].

Science.gov (United States)

Yan, Guang; Zhao, Su-ling; Xu, Zheng; Zhang, Fu-jun; Kong, Chao; Liu, Xiao-dong; Gong, Wei; Gao, Li-yan

2011-07-01

Series of organic light emitting devices with basic structure of ITO/PCBM: PVK(x Wt%, approximately 40 nm)/DPVBi(30 nm)/Alq3 (30 nm)/Al were fabricated in order to investigate the carrier recombination region movement in these devices. The carrier injection-dependent, the carrier transport-dependent and the voltage-dependent carrier recombination region movements were investigated respectively by modifying cathode with lithium fluoride, by changing the doping concentration of PCBM and by changing the voltage on the devices. The physical mechanism behind the voltage-dependent carrier recombination region movement was discussed.

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase.

Science.gov (United States)

Shang, Pengcheng; Misra, Saurav; Hause, Ben; Fang, Ying

2017-07-15

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3C pro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event. IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we

A comparative study of natural 40K content estimated through whole body counting and dietary intake around Narora Atomic Power Station

International Nuclear Information System (INIS)

Gautam, Y.P.; Dube, B.; Hegde, A.G.

2005-01-01

773 radiation workers at NAPS, aged 20 to 59 years, were monitored using Shadow Shield Whole Body Counting System having NaI (Tl) crystal coupled with NETS-3 1 K Multi Channel Analyser (MCA) to determine the 40 K activity in the body and assess internal dose due to naturally occurring 40 K. The data have been segregated to make analyses for vegetarian and non-vegetarian. The average annual dose from 40 K for the subjects is evaluated as 156.4 ± 36.1 mSv. Natural 40 K content in 463 environmental samples collected from Narora environ estimated using NaI(Tl) well type detector coupled with 1 K NETS-3 Multichannel analyser (MCA). Assessment of daily intake of natural 40 K has been estimated from average daily intake of dietary items and the associated 40 K activity. It works out to be 67.17 ± 16.28 Bq/d and that obtained through analysis of complete meal samples was 73.22 ± 9.78 Bq/ d. The average annual dose to a member of public of this region due to natural 40 K through ingestion route works out to be 152.12 ± 36.83 mSv/year. (author)

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

Directory of Open Access Journals (Sweden)

Remy Froissart

2005-03-01

Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Directory of Open Access Journals (Sweden)

S.C. Silva

2010-02-01

Full Text Available Bovine herpesvirus 5 (BoHV-5, the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99. The recombinants are defective in glycoprotein E (BoHV-5gEΔ, thymidine kinase (BoHV-5TKΔ and both proteins (BoHV-5gEΔTKΔ. Rabbits inoculated with the parental virus (N = 8 developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi. Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8 or BoHV-5gEΔTKΔ (N = 8 remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

The typical RB76 recombination breakpoint of the invasive recombinant tomato yellow leaf curl virus of Morocco can be generated experimentally but is not positively selected in tomato.

Science.gov (United States)

Belabess, Z; Urbino, C; Granier, M; Tahiri, A; Blenzar, A; Peterschmitt, M

2018-01-02

TYLCV-IS76 is an unusual recombinant between the highly recombinogenic tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV), two Mediterranean begomoviruses (Geminiviridae). In contrast with the previously reported TYLCV/TYLCSV recombinants, it has a TYLCSV derived fragment of only 76 nucleotides, and has replaced its parental viruses in natural conditions (Morocco, Souss region). The viral population shift coincided with the deployment of the popular Ty-1 resistant tomato cultivars, and according to experimental studies, has been driven by a strong positive selection in such resistant plants. However, although Ty-1 cultivars were extensively used in Mediterranean countries, TYLCV-IS76 was not reported outside Morocco. This, in combination with its unusual recombination pattern suggests that it was generated through a rare and possibly multistep process. The potential generation of a recombination breakpoint (RB) at locus 76 (RB76) was investigated over time in 10 Ty-1 resistant and 10 nearly isogenic susceptible tomato plants co-inoculated with TYLCV and TYLCSV clones. RB76 could not be detected in the recombinant progeny using the standard PCR/sequencing approach that was previously designed to monitor the emergence of TYLCV-IS76 in Morocco. Using a more sensitive PCR test, RB76 was detected in one resistant and five susceptible plants. The results are consistent with a very low intra-plant frequency of RB76 bearing recombinants throughout the test and support the hypothesis of a rare emergence of TYLCV-IS76. More generally, RBs were more scattered in resistant than in susceptible plants and an unusual RB at position 141 (RB141) was positively selected in the resistant cultivar; interestingly, RB141 bearing recombinants were detected in resistant tomato plants from the field. Scenarios of TYLCV-IS76 pre-emergence are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

Temperature-dependent measurement of Auger recombination in In{sub 0.40}Ga{sub 0.60}N/GaN red-emitting (λ = 630 nm) quantum dots

Energy Technology Data Exchange (ETDEWEB)

Frost, Thomas; Banerjee, Animesh; Jahangir, Shafat; Bhattacharya, Pallab, E-mail: pkb@eecs.umich.edu [Center for Photonics and Multiscale Nanomaterials, Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, Michigan 48109-2122 (United States)

2014-02-24

We have derived the Auger recombination coefficients, as a function of temperature, for In{sub 0.4}Ga{sub 0.6}N/GaN self-organized quantum dots from large-signal modulation measurements made on lasers in which the quantum dots form the gain media. The value of C{sub a} = 1.3 ±0.2 × 10{sup −31} cm{sup 6} s{sup −1} at room temperature and the coefficient decreases with increase of temperature.

Finanční deriváty v České republice a ve světě

OpenAIRE

Petrov, Ondřej

2011-01-01

This thesis deals with the financial derivatives and trading in the world and the Czech Republic. The introductory part defines the basic concepts associated with financial derivatives, describes their types and method of statistical reporting. Briefly outlines the basic types of derivatives and their history. The second part evaluates the derivatives trading on Exchange and OTC markets. It is primarily focused on the latest development during the financial crisis. The third part examines mor...

Platelet-Rich Blood Derivatives for Stem Cell-Based Tissue Engineering and Regeneration

NARCIS (Netherlands)

Masoudi, E.A.; Ribas, J.; Kaushik, G.; Leijten, Jeroen Christianus Hermanus; Khademhosseini, A.

2016-01-01

Platelet-rich blood derivatives have been widely used in different fields of medicine and stem cell-based tissue engineering. They represent natural cocktails of autologous growth factors, which could provide an alternative for recombinant protein-based approaches. Platelet-rich blood derivatives,

Cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the MVP N termini and VPARP.

Science.gov (United States)

Mikyas, Yeshi; Makabi, Miriam; Raval-Fernandes, Sujna; Harrington, Lea; Kickhoefer, Valerie A; Rome, Leonard H; Stewart, Phoebe L

2004-11-12

The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large beta-propeller fold.

Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

Science.gov (United States)

Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

2013-09-01

In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Meiotic Recombination

Energy Technology Data Exchange (ETDEWEB)

Gregory p. Copenhaver

2011-11-09

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

40 CFR 721.10193 - 1-Butanaminium, N-(3-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Butanaminium, N-(3-aminopropyl)-N...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. (a) Chemical substance and...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts (PMN P-06-263, Chemical B; CAS No...

Sensitization of recombinant human tumor necrosis factor-related apoptosis-inducing ligand-resistant malignant melanomas by quercetin.

Science.gov (United States)

Turner, Katherine A; Manouchehri, Jasmine M; Kalafatis, Michael

2018-03-28

Malignant melanoma is the most commonly diagnosed skin cancer associated with a high rate of metastasis. Low-stage melanoma is easily treated, but metastatic malignant melanoma is an extremely treatment-resistant malignancy with low survival rates. The application of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) for the treatment of metastatic malignant melanoma holds considerable promise because of its selective proapoptotic activity towards cancer cells and not nontransformed cells. Unfortunately, the clinical utilization of rhTRAIL has been terminated due to the resistance of many cancer cells to undergo apoptosis in response to rhTRAIL. However, rhTRAIL-resistance can be abrogated through the cotreatment with compounds derived from 'Mother Nature' such as quercetin that can modulate cellular components responsible for rhTRAIL-resistance. Here, we show that rhTRAIL-resistant malignant melanomas are sensitized by quercetin. Quercetin action is manifested by the upregulation of rhTRAIL-binding receptors DR4 and DR5 on the surface of cancer cells and by increased rate of the proteasome-mediated degradation of the antiapoptotic protein FLIP. Our data provide for a new efficient and nontoxic treatment of malignant melanoma.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors.

Science.gov (United States)

Yamashita, Toshiharu; Okura, Masae; Ishii-Osai, Yasue; Hida, Tokimasa

2016-10-01

Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP-A to -G, and a variant type (XP-V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP-A, -B, -C, -D, -F or -G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV-C at 5-20 J/m 2 was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP-E and XP-V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co-infection of Ad-XPA with Ad-XPE increased survival rate of XP-E cells after UV-C exposure. When XP-V cell strains, including one derived from a Japanese patient, were infected with Ad-XPV, exposed to UV-B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP-V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP-E and XP-V. © 2016 Japanese Dermatological Association.

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus

DEFF Research Database (Denmark)

Langeveld, J.P.M.; Brennan, F.R.; Martinez-Torrecuadrada, J.L.

2001-01-01

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1...

Optical emissions from the dissociative recombination of N{sub 2}H{sup +}, HCO{sup +}, HOC{sup +}, and HNC{sup +}

Energy Technology Data Exchange (ETDEWEB)

Johnsen, R [Department of Physics and Astronomy University of Pittsburgh, Pittsburgh, PA 15260 (United States); Golde, M F [Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Rosati, R E [Smithsonian Astrophysical Observatory, 60 Garden St. MS-50 Cambridge, MA, 02138 (United States); Pappas, D [Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD (United States); Skrzypkowski, M P, E-mail: rj@pitt.ed [Prometheus Energy Company, 3311 S. 120th Place Suite 100, Seattle, WA 98168 (United States)

2009-11-15

We present recent flowing-afterglow measurements of branching fractions for electronically and vibrationally excited products arising from the dissociative recombination of N{sub 2}H{sup +}, HCO{sup +}, HOC{sup +}, and HNC{sup +} ions with thermal electrons. State-specific yields were derived by fitting the observed, spatially resolved emission band intensities to models that simulate all ion-chemical processes, recombination, diffusion, and gas mixing.

Occupational exposure to the personnel regarded by NRB-76/87 to B category

Energy Technology Data Exchange (ETDEWEB)

Gubatova, D; Balode, G; Nemiro, E [Medical Institute, Riga (USSR)

1990-01-01

During the decades use of radioactive sources in all branches of national economy has increased. Due to this tendency the number of exposed individuals from artificial sources including those exposed only occasionally (the so-called B category) increases. For this category the personnel dosimetry is not obligatory, and radioactive monitoring of their working and living places would do. Nevertheless, the thermoluminescent personnel monitoring data derived by us show that occupational exposure of this category is near, and occasionally even higher than B category exposure limit. So, for example, anesthesiologists annual exposure makes 4.5 mSv, that of surgeons 6.5 mSv, of anesthetic nurses 4.0 mSv (Traumatological and Orthopedic Institute personnel). Annual exposure of 'Medtekhnika' factory electromechanics ought to repair, set up and check up X-ray devices in 1987 was 5 mSv. (author).

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination▿

Science.gov (United States)

Taucher, Christian; Berger, Angelika; Mandl, Christian W.

2010-01-01

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses

Derived intervention levels for radionuclides in foods in Chile

International Nuclear Information System (INIS)

Pinones O, O.; Tomicic M, I.

1992-01-01

The present paper reports the methodology and Derived Intervention Levels (DILs) for radionuclides in foods in Chile for international trading, in the event of a nuclear accident or radiological emergency. The radionuclides of interest were classified in three groups, according to their radiotoxicity, and the DILs calculated for each category of food (Cereals, Roots, Vegetables, Fruits, Meat, Fish and Milk). Values of 1[mSv.y -1 ] [5] as a Dose Reference Level and 507 [Kg.a -1 ] [1] as a Total Annual Consumption, were considered. (author)

Alkyl-fluorinated thymidine derivatives for imaging cell proliferation

International Nuclear Information System (INIS)

Toyohara, Jun; Hayashi, Akio; Gogami, Akie; Hamada, Masahiro; Hamashima, Yoshio; Katoh, Takahiro; Node, Manabu; Fujibayashi, Yasuhisa

2006-01-01

Derivatives of 2'-deoxyuridine that contain fluoroalkyl groups at the C5 position and derivatives of thymidine that contain fluoroalkyl groups at the N3 position were synthesized and examined in three in vitro assays designed to evaluate their potential as radiopharmaceuticals for imaging cellular proliferation. Three of the former nucleosides and five of the latter were synthesized. The three assays were as follows: (a) phosphoryl transfer assay, which showed that all three of the former nucleosides and four of the latter ones were phosphorylated by recombinant human thymidine kinase 1 (TK1) and that N 3 -(2-fluoroethyl)-thymidine (NFT202) was the most potent substrate of the eight nucleosides studied; (b) transport assay, which indicated that all eight nucleosides had good affinity for an 6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine-sensitive mouse erythrocyte nucleoside transporter, with inhibition constants in the range of 0.02-0.55 mM; and (c) degradation assay, which showed that all but one of the former nucleosides and none of the latter were degraded by recombinant Escherichia coli thymidine phosphorylase (an enzyme that catalyzes the glycosidic bond of thymidine and 2'-deoxyuridine derivatives). From these in vitro screening assays, we selected NFT202 as a candidate for subsequent in vivo evaluation because this compound met the three minimum requirements of the in vitro screening assays and had the most potent phosphorylation activity as a substrate for recombinant human TK1

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

Directory of Open Access Journals (Sweden)

Willson Richard C

2010-12-01

Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use

Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum.

Science.gov (United States)

Speranskaya, Anna S; Krinitsina, Anastasia A; Kudryavtseva, Anna V; Poltronieri, Palmiro; Santino, Angelo; Oparina, Nina Y; Dmitriev, Alexey A; Belenikin, Maxim S; Guseva, Marina A; Shevelev, Alexei B

2012-08-01

The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Evaluation for committed effective dose due to dietary foods by the intake for Japanese adults

International Nuclear Information System (INIS)

Sanada, Tetsuya

2011-01-01

Explained are the internal exposure (IE) in Japanese adults due to the ordinary intake of food and the committed effective dose (CED) derived from it, which can be the basic reference data for estimating CED at emergency like the recent Fukushima Disaster. IE lasts as long as radioactive substances exist in the body and its health risk is assessed throughout the lifetime, 50 years in adult and in children until their age of 70 years, by CED based on consideration of the physical and biological halftimes of radionuclides involved. CED (mSv) is calculated by an equation given in International Commission of Radiological Protection (ICRP) Pub. 72, using radioactivity level (Bq/kg), intake (kg/y) and age-dependent dose coefficient (mSv/Bq). Japan MEXT in its homepage (http://www.kankyo-hoshano.go.jp/) publishes the states of environmental radioactivity levels including those of daily Japanese food. The food survey of yearly changes of radioactivity level (Bq/man/day) indicates that, when compared with the level in 2009, it is two times higher in 1970s due to old in-atmospheric nuclear experiments, and is decreased gradually with a temporary peak around 1986 by Chernobyl accident. Studies on certain natural and artificial radioisotopes in 240 kinds of current food in 1989-2005 reveal that Po-210 level in fishes and shellfishes is particularly higher than others like St-90, Cs-137, Rd-226, Pb-210 and so on. CED in adults is calculated from yearly intake of foods (kg/y), their radioactivity (Bq/y) and the coefficient above, to be 0.80 mSv, and 0.98 mSv when the contribution of K-40 is taken in calculation. The proportion of the contribution in the latter estimated CED accounts for 74% (0.73 mSv) by Po-210 and for 18% (0.18 mSv) by K-40, which is conceivably derived from intake of more seafood by Japanese than other people in the world (average 0.070 and 0.17 mSv, respectively). (T.T.)

Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster.

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Piontkivska, Helen; Matos, Luis F; Paul, Sinu; Scharfenberg, Brian; Farmerie, William G; Miyamoto, Michael M; Wayne, Marta L

2016-10-05

Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster. Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. Here we use SOLiD TM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. We also find evidence of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e., rare); others were shared by four or even all five of the flies (i.e., common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster

Science.gov (United States)

Piontkivska, Helen; Matos, Luis F.; Paul, Sinu; Scharfenberg, Brian; Farmerie, William G.; Miyamoto, Michael M.; Wayne, Marta L.

2016-01-01

Abstract Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster. Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. Here we use SOLiDTM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be “resistant” if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. We also find evidence of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e., rare); others were shared by four or even all five of the flies (i.e., common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts. PMID:27614234

Production of biologically active recombinant human factor H in Physcomitrella.

Science.gov (United States)

Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

2011-04-01

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Protection by recombinant viral proteins against a respiratory challenge with virulent avian metapneumovirus.

Science.gov (United States)

Chary, Parag; Njenga, M Kariuki; Sharma, Jagdev M

2005-12-15

Protection by recombinant avian metapneumovirus (aMPV) N or M proteins against a respiratory challenge with virulent aMPV was examined. N, M or N+M proteins were administered intramuscularly (IM) with incomplete Freund's adjuvant (IFA) or by the oculonasal (ON) route with cholera toxin-B (CTB). Each turkey received 40 or 80 microg of each recombinant protein. Birds were considered protected against challenge if the challenge virus was not detectable in the choanal swabs by RT-PCR. At a dose of 40 microg/bird, N protein given with IFA by the IM route protected eight out of nine birds. M protein at the same dose protected three out of seven birds, while a combination of N+M proteins (40 microg each) protected three out of four birds. At a dose of 80 microg of each of N and M proteins per bird given with IFA by the IM route, 100% protection was achieved. ON immunization with a mixture of N and M proteins induced partial protection when the proteins were given with CTB; no detectable protection was noted without CTB. N and M proteins induced anti-aMPV antibodies, although protection against virulent virus challenge did not appear to be associated with the level or presence of antibodies.

Evolution of cagA oncogene of Helicobacter pylori through recombination.

Directory of Open Access Journals (Sweden)

Yoshikazu Furuta

Full Text Available Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i homologous recombination between DNA sequences for CagA multimerization (CM sequence; (ii recombination between DNA sequences for the EPIYA motif; and (iii recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

International Nuclear Information System (INIS)

Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

2012-01-01

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

Energy Technology Data Exchange (ETDEWEB)

Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Experimental evolution of recombination and crossover interference in Drosophila caused by directional selection for stress-related traits.

Science.gov (United States)

Aggarwal, Dau Dayal; Rashkovetsky, Eugenia; Michalak, Pawel; Cohen, Irit; Ronin, Yefim; Zhou, Dan; Haddad, Gabriel G; Korol, Abraham B

2015-11-27

Population genetics predicts that tight linkage between new and/or pre-existing beneficial and deleterious alleles should decrease the efficiency of natural selection in finite populations. By decoupling beneficial and deleterious alleles and facilitating the combination of beneficial alleles, recombination accelerates the formation of high-fitness genotypes. This may impose indirect selection for increased recombination. Despite the progress in theoretical understanding, interplay between recombination and selection remains a controversial issue in evolutionary biology. Even less satisfactory is the situation with crossover interference, which is a deviation of double-crossover frequency in a pair of adjacent intervals from the product of recombination rates in the two intervals expected on the assumption of crossover independence. Here, we report substantial changes in recombination and interference in three long-term directional selection experiments with Drosophila melanogaster: for desiccation (~50 generations), hypoxia, and hyperoxia tolerance (>200 generations each). For all three experiments, we found a high interval-specific increase of recombination frequencies in selection lines (up to 40-50% per interval) compared to the control lines. We also discovered a profound effect of selection on interference as expressed by an increased frequency of double crossovers in selection lines. Our results show that changes in interference are not necessarily coupled with increased recombination. Our results support the theoretical predictions that adaptation to a new environment can promote evolution toward higher recombination. Moreover, this is the first evidence of selection for different recombination-unrelated traits potentially leading, not only to evolution toward increased crossover rates, but also to changes in crossover interference, one of the fundamental features of recombination.

Partial radiative-recombination cross sections for excited states of hydrogen

International Nuclear Information System (INIS)

Fazio, P.M.; Copeland, G.E.

1985-01-01

The squares of the dipole and quadrupole matrix elements for the free-to-bound transitions of hydrogen up to bound states Vertical Barn = 20,l = 19> are derived in closed analytic form as a function of the kinetic energy of the free electron. Coulomb wave functions are used for the free as well as the bound states and, thus, the results are good for any electron energy. Several interesting effects are found. First, the transition probabilities are maximum for recombination into specific intermediate-angular-momentum states at low energies (w<1 eV) and where the free-state angular momentum is greater than that of the bound state. Further, that specific intermediate-angular-momentum state depends on the kinetic energy of the free electron. This behavior is in contrast to the ''normal'' behavior of the transition strengths where recombination into s states is greatest and decreases with increasing angular momentum. Second, the quadrupole matrix elements vanish for certain velocities of the free electron. These ''zeros'' produce minima in the corresponding quadrupole cross sections. Finally, the calculated partial cross sections for recombination into high-angular-momentum states are greater when quadrupole transitions are included

Recombination of cluster ions

Science.gov (United States)

Johnsen, Rainer

1993-01-01

Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

Biomimetic calcium phosphate coatings on recombinant spider silk fibres

Energy Technology Data Exchange (ETDEWEB)

Yang Liang; Habibovic, Pamela; Van Blitterswijk, Clemens A [Department of Tissue Regeneration, University of Twente, PO Box 217, 7500 AE Enschede (Netherlands); Hedhammar, My; Johansson, Jan [Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, the Biomedical Centre, Box 575, 751 23 Uppsala (Sweden); Blom, Tobias; Leifer, Klaus [Department of Engineering Sciences, Uppsala University, Box 534, S-751 21 Uppsala (Sweden)

2010-08-01

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs.

Biomimetic calcium phosphate coatings on recombinant spider silk fibres

International Nuclear Information System (INIS)

Yang Liang; Habibovic, Pamela; Van Blitterswijk, Clemens A; Hedhammar, My; Johansson, Jan; Blom, Tobias; Leifer, Klaus

2010-01-01

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs.

QTL analysis of seed dormancy in Arabidopsis using recombinant inbred lines and MQM mapping

NARCIS (Netherlands)

Schaar, Wybe van der; Alonso-Blanco, Carlos; Léon-Kloosterziel, Karen M.; Jansen, Ritsert C.; Ooijen, Johan W. van; Koornneef, Maarten

1997-01-01

The genetic differences for seed germination between two commonly used Arabidopsis thaliana ecotypes Ler and Col, both showing a low level of seed dormancy, were investigated. The analysis was performed with 98 recombinant inbred lines (RILs) derived from the cross between the two ecotypes, and

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.

Science.gov (United States)

Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

2007-01-01

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.