Third international challenge to model the medium- to long-range transport of radioxenon to four Comprehensive Nuclear-Test-Ban Treaty monitoring stations.

In 2015 and 2016, atmospheric transport modeling challenges were conducted in the context of the Comprehensive Nuclear-Test-Ban Treaty (CTBT) verification, however, with a more limited scope with respect to emission inventories, simulation period and number of relevant samples (i.e., those above the Minimum Detectable Concentration (MDC)) involved. Therefore, a more comprehensive atmospheric transport modeling challenge was organized in 2019. Stack release data of Xe-133 were provided by the Institut National des Radioéléments/IRE (Belgium) and the Canadian Nuclear Laboratories/CNL (Canada) and accounted for in the simulations over a three (mandatory) or six (optional) months period. Best estimate emissions of additional facilities (radiopharmaceutical production and nuclear research facilities, commercial reactors or relevant research reactors) of the Northern Hemisphere were included as well. Model results were compared with observed atmospheric activity concentrations at four International Monitoring System (IMS) stations located in Europe and North America with overall considerable influence of IRE and/or CNL emissions for evaluation of the participants' runs. Participants were prompted to work with controlled and harmonized model set-ups to make runs more comparable, but also to increase diversity. It was found that using the stack emissions of IRE and CNL with daily resolution does not lead to better results than disaggregating annual emissions of these two facilities taken from the literature if an overall score for all stations covering all valid observed samples is considered. A moderate benefit of roughly 10% is visible in statistical scores for samples influenced by IRE and/or CNL to at least 50% and there can be considerable benefit for individual samples. Effects of transport errors, not properly characterized remaining emitters and long IMS sampling times (12-24 h) undoubtedly are in contrast to and reduce the benefit of high-quality IRE and CNL stack data. Complementary best estimates for remaining emitters push the scores up by 18% compared to just considering IRE and CNL emissions alone. Despite the efforts undertaken the full multi-model ensemble built is highly redundant. An ensemble based on a few arbitrary runs is sufficient to model the Xe-133 background at the stations investigated. The effective ensemble size is below five. An optimized ensemble at each station has on average slightly higher skill compared to the full ensemble. However, the improvement (maximum of 20% and minimum of 3% in RMSE) in skill is likely being too small for being exploited for an independent period.

Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings.

SH-EP is the major papain-type proteinase expressed in cotyledons of germinated Vigna mungo seeds. The proteinase possesses a KDEL sequence at the C-terminus although the mature form of SH-EP is localized in vacuoles. It has also been shown that the proform of SH-EP is accumulated at the edge or middle region of the endoplasmic reticulum, and the accumulated proSH-EP is directly transported to vacuoles via the KDEL-tailed cysteine proteinase-accumulating vesicle, KV. In this study, to address the transport machinery of proSH-EP through KV, putative receptor for proSH-EP was isolated from membrane proteins of cotyledons of V. mungo seedlings using a proSH-EP-immobilized column. The deduced amino acid sequence from cDNA to the protein revealed that the putative receptor for proSH-EP is a member of vacuolar sorting receptor, VSR, that is known to be localized in the Golgi-complex and/or clathrin coated vesicle. We carried out subcellular fractionation of cotyledon cells and subsequently conducted SDS-PAGE/immunoblotting and immunocytochemistry with anti-V. mungo VSR (VmVSR) or SH-EP antibody. The results showed that VmVSR is co-localized in the fraction of the gradient in which KV existed.

NSF is required for the brefeldin A-promoted disassembly of the Golgi apparatus.

N-Ethylmaleimide-sensitive factor (NSF) is required for multiple pathways of vesicle-mediated protein transport. Microinjection of a monoclonal anti-NSF antibody almost completely blocked brefeldin A-promoted Golgi disassembly without affecting the rapid release of beta-COP, a subunit of the Golgi coat proteins (COPI), from the Golgi apparatus. Similar results were obtained using a dominant-negative NSF which is known to compete with endogenous NSF. The present results suggest that an NSF-mediated step is present in the brefeldin A-promoted disassembly of the Golgi apparatus.

Possible involvement of heterotrimeric G proteins in the organization of the Golgi apparatus.

Nordihydroguaiaretic acid (NDGA) caused disassembly of the Golgi apparatus of NRK cells in a dose-, time-, and energy-dependent manner but not in a microtubule-dependent manner. In contrast to brefeldin A, NDGA did not cause release of beta-COP, a component of Golgi-derived vesicles. However, NDGA-induced disassembly was blocked by AlF4-, an activator of the heterotrimeric but not the small GTP-binding proteins. In digitonin-permeabilized cells, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) as well as AlF4- blocked the NDGA-promoted disassembly of the Golgi apparatus, and Gbetagamma (betagamma subunits of heterotrimeric G proteins) reversed this effect. Our present results suggest the possible involvement of heterotrimeric G proteins in the organization of the Golgi apparatus.

SNAP prevents Mg(2+)-ATP-induced release of N-ethylmaleimide-sensitive factor from the Golgi apparatus in digitonin-permeabilized PC12 cells.

The N-ethylmaleimide-sensitive factor (NSF), which is involved in the multisteps of protein transport, is released from Golgi membranes on in vitro incubation with Mg(2+)-ATP. However, several lines of evidence suggest that NSF is associated with membranes in spite of the presence of Mg2+ and ATP in vivo. We have used digitonin-permeabilized PC12 cells to investigate the mechanism underlying the association of NSF with membranes. In PC12 cells, immunoreactivity for NSF was observed in the nuclear membranes, the Golgi apparatus, and neuronal growth cones, where synaptic vesicles are concentrated. NSF associated with the Golgi apparatus was released on incubation with Mg(2+)-ATP, whereas NSF in the nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg(2+)-ATP-induced release of NSF from the Golgi apparatus. Chromatographic analyses revealed that the factor(s) that prevents NSF release from the Golgi apparatus was eluted at the same position as the soluble NSF attachment proteins (SNAPs). Purified His6-tagged alpha-SNAP exhibited such activity. His6-tagged alpha-SNAP also prevented the Mg(2+)-ATP-induced release of NSF from isolated Golgi membranes.

Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer.

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.

Changing the spacing between metal-binding motifs decreases stability and transforming activity of the human papillomavirus type 18 E7 oncoprotein.

Spacing composed of 29 amino acids (AAs) between a pair of metal-binding motifs, Cys-X-X-Cys, is common to human papillomavirus (HPV) zinc-binding oncoproteins E6 and E7 from various HPV types. From the HPV 18 E7 gene encoding a 105-AA protein with the motifs at AAs 65-68 and 98-101, we constructed expression plasmids for two mutants, 18del73 and 18ins84, with varied spacing between the motifs. Mutant proteins 18del73 and 18ins84 had a deletion from AAs 74 to 88 and an insertion of three AAs substituting for AA 85, respectively. The mutations lowered the efficiency of E7-mediated focal transformation of rat 3Y1 cells, approximately parallel to the reduced level of steady-state E7 expressed in COS-1 cells. It is likely that, besides the presence of the motifs, the conserved spacing between the motifs is important for a stable structure of E7, but is not essential for the E7-transforming activity.

Unusual structure of plasmid DNA formed in transformants of Escherichia coli.

An unusual structural component of plasmid pUC18 was observed in transformants of Escherichia coli strains. This molecule migrated faster than superhelical topoisomers in agarose gel electrophoresis and was difficult to stain with ethidium bromide. This unusual topology appeared independently of gyrase mutation, and the recA genotype may relate to accumulation of the plasmids conforming to the unusual topology.

Topological alteration of plasmid DNA during cell growth of Escherichia coli.

Superhelical alteration of plasmid DNA in Escherichia coli cells was observed during cell growth. Plasmid DNAs were being supercoiled while the cells were actively dividing, and they were partially relaxed after the cultures reached stationary. Unknown structural materials of plasmid DNA which migrated faster than usual superhelices in agarose gel electrophoresis were found among the strains regardless of gyrase genotype.

Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif.

The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136, and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. Immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-66 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 cells was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-66 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 16 E7 transformation of rat 3Y1 cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6.

Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells.

The human papillomavirus type 16 E7 protein is a 98-amino acid (AA)-long nuclear oncoprotein. We established eight independent mouse hybridoma cell lines producing monoclonal antibodies (MAbs) specific for the E7 protein and characterized the MAbs by competitive binding analysis with the bacterially expressed, nonfusion E7 protein and synthesized oligopeptides. The MAbs were classified into two groups; three and five MAbs recognizing epitopes in probable immunodominant regions AAs 8 to 22 (region I) and AAs 39 to 54 (region II), respectively. These MAbs were capable of immunoprecipitating the E7 protein transiently expressed in monkey COS-1 cells. By immunofluorescence staining of the acetone-fixed E7-producing COS-1 cells, the MAbs for regions I and II detected no E7 protein and only cytoplasmic E7 protein, respectively, whereas a rabbit polyclonal antiserum (anti-lac-E7) detects both nuclear and cytoplasmic E7 proteins. Like anti-lac-E7, the MAbs of the two groups stained both nuclear and cytoplasmic E7 proteins in the COS-1 cells that were denatured with formaldehyde. The results show that two immunodominant regions of the HPV 16 E7 protein are masked in the COS-1 nuclei for binding with the corresponding MAbs. The masking of the intranuclear E7 protein may result from complex formation with cellular proteins (or structures), polymerization, or self-folding.

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.

Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein.

Human papillomavirus type 16 E7 protein, expressed in Escherichia coli as fusion protein lac alpha peptide-E7 (lac-E7), was purified by electrophoresis and used to immunize rabbits to raise antisera. The anti-lac-E7 sera recognized another bacterially expressed fusion protein trpE-E7 by the Western blot method. The antisera were used to identify E7 protein transiently expressed in monkey COS-1 cells from an SV40-derived expression plasmid containing E7 gene. Like the E7 protein from CaSki cells, the majority of 19K E7 protein in the transfected COS-1 cells was found by immunoprecipitation in the soluble cytoplasmic fraction. By immunofluorescence staining, however, the E7 protein was detectable in the nuclei of the transfected COS-1 cells. The results suggest that the E7 protein expressed in monkey cells is nuclear, although it is readily released from nuclei when the cell structure is broken.

Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) open reading frames (ORF) E6, E7, and E6E7, placed under the control of dexamethasone-inducible mouse mammary tumor virus long terminal repeat, were introduced into rat 3Y1 cells, an immortalized fibroblast line, with the aid of neomycin-selection. The cell clones containing inducible HPV 16 ORFs were selected and examined for DNA synthesis. Following induction of HPV mRNA synthesis by the hormone, DNA synthesis was stimulated in the cells containing E7 or E6E7 ORFs. The data indicate that expression of the HPV 16 E7 gene is mitogenic for rat 3Y1 cells.

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.

Effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by sodium chromate (VI).

The effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by Na2CrO4 were examined by alkaline elution. Incubation of Chinese hamster V-79 cells with alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure to Na2CrO4 resulted in a decrease of DNA breaks produced by this compound. However, similar pretreatment with riboflavin (vitamin B2) or Na2SeO3 resulted in an enhanced formation of breaks induced by Na2CrO4. Pretreatment with Na2SeO3 resulted in increased levels of glutathione in these cells while levels of glutathione remained the same with vitamin E or vitamin B2. These results suggest that Na2CrO4 induced DNA breaks appear to be mediated by the formation of free radicals and/or cellular reductive metabolism.

Mutation in the VP-1 gene is responsible for the extended host range of a monkey B-lymphotropic papovavirus mutant capable of growing in T-lymphoblastoid cells.

Monkey B-lymphotropic papovavirus (LPV) replicates only in B-lymphoblastoid cells, whereas the LPV mutant 76 (LPV-76) can grow in either B- or T-lymphoblastoid cells. The nucleotide sequence of the wild-type LPV PstI B segment was compared with that of LPV-76 PstI-B, within which the mutation responsible for the extended host range had been located. The VP-1 coding region of LPV-76 was found to have mutations, single-base substitutions causing three amino acid substitutions.

Monkey B-lymphotropic papovavirus genome: the entire DNA sequence and variable regions.

DNA from monkey B-lymphotropic papovavirus (LPV) adapted to growth in human B-lymphoblastoid cell line BJA-B consisted of three classes of molecules of 5.1, 5.0, and 4.9 kilobases (kb), and the entire DNA sequence of 5.1-kb molecule was determined. The three types of LPV DNA yielded infectious virus upon transfection to BJA-B cells. Comparison of the structures among these clones and clone K38 sequenced by Pawlita et al. (1985) suggests that the transcriptional control region, the small-T antigen gene, and the VP-1 gene are variable in the LPV genome, and that the 5.0-kb LPV resembles the parent virus, from which all the others had evolved by duplication and deletion during passages in BJA-B cells, probably for adaptation to better growth in human cells.