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At least 1,027 records · Page 57Linked to original sources
PubMed2000

Mapping the inter-RNA interaction of bacterial virus phi29 packaging RNA by site-specific photoaffinity cross-linking.

During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure.

Bacillus Phages↗
PubMed2005

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor.

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.

Adenosine Triphosphatases↗
PubMedDate not supplied

Migration into food of polyethylene terephthalate (PET) cyclic oligomers from PET microwave susceptor packaging.

A quantitative method has been developed to measure the migration of polyethylene terephthalate (PET) cyclic oligomers from aluminized PET susceptor film-type food packaging into several food types. Microwaveable French fries, popcorn, fish sticks, waffles and pizza sold in susceptor-type packaging were purchased in local markets, cooked according to package instructions and analysed for PET oligomers. Appropriate food blanks were cooked in glass containers. Quantities of PET oligomers found in the foods ranged from less than 0.012 micrograms/g to approximately 7 micrograms/g.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

1H-NMR for the safety control of food packaging materials: analysis of extracts from polyolefin samples.

A new approach for the identification of the additives in extracts of packaging plastics, using proton nuclear magnetic resonance (1H-NMR), is presented. The technique can be used in a preliminary step for the determination of contaminants potentially released by the food packaging materials; it may greatly reduce the time required to identify the constituents of the materials, either individually or as functional classes. A classification of the EEC additives is proposed on the basis of chemical shift. 1H-NMR can also be used as a fingerprint technique in the quality control of the food packaging materials.

Chromatography↗
PubMedDate not supplied

Safety assessment of microwave susceptors and other high temperature packaging materials.

A number of packaging materials are being used not only to contain food during distribution but also to serve as the cooking container. The higher temperatures that these materials reach led the US Food and Drug Administration (FDA) to issue an intent to publish new regulations in 1989. The food and packaging industries responded by conducting extensive research and submitting the results to FDA. The methods used and results obtained are discussed. Most of the data were focused on microwave susceptors and the volatile compounds generated. One project showed that for a specific product, popcorn, there was no transfer into the food. Work is continuing to validate methods to test for non-volatile compounds. In addition to susceptors, various paper and plastic materials are used in dual ovenable (microwave and conventional ovens) applications. Most of the research on these materials has investigated the food contact temperatures on testing for migrants. An update on the current regulatory status of packaging materials intended for high temperature use in the US is discussed.

Consumer Product Safety↗
PubMedDate not supplied

The role of the Scientific Committee for Food in evaluating plastics for packaging.

One of the tasks of the Scientific Committee for Food (SCF) is to advise the Commission of the European Communities (CEC) on the safety-in-use of monomers, other starting substances and additives used in food packaging materials. This advice forms the basis of Community Directives for the regulation of food packaging materials by a system of positive lists of substances authorized for use. The SCF considers the available migration and toxicity data and classifies each substance into one of ten lists, reflecting whether or not there are adequate data and whether the data indicate the potential for adverse effects. This paper describes the SCF classification system and discusses the rationale behind the SCF approach to toxicity testing and evaluation of food packaging materials, with particular emphasis on the recent change which took place in 1990 when the Committee issued a new set of guidelines. These guidelines outlined a new tiered approach to toxicity testing, based on the principle that the greater the potential human exposure to a substance, the more toxicity data are required to make a sound health assessment.

Animals↗
PubMed1981

Requirements for food packaged in polymeric films.

The requirements of barrier properties of packaging materials against environmental factors vary with food products. The chemical, physical, and biological mechanisms of food deterioration due to environmental factors, vital properties required in packaging materials, and developments in progress and future trends to maintain the required standard of food quality have been critically reviewed. Theoretical and experimental results for a variety of food products in relation to the properties of the packaging materials are discussed. Methods of prediction of food stability and their industrial applications are also emphasized by specific examples.

Cellophane↗
PubMed2002

Audit as an educational package for family medicine residents in primary health care (PHC) rotation.

BACKGROUND: The Saudi Board of Family and Community Medicine requires residents of family medicine to perform an audit during PHC rotation. The aim of the project is to introduce the residents to their future responsibilities towards improving health services in PHC setting. THE PACKAGE: A series of small group discussion sessions introduces the concept of audit to residents. Each of them is then asked to audit one aspect of his work while in the rotation. The resident is assigned to a facilitator (assessor). At the end of the project the resident has to submit his work in a predetermined format. This format aims at helping the resident to comprehend his work, be concise and help the assessor to evaluate the work with considerable ease. RESULTS: The package is evaluated through regular opinion surveys that provoke participants' satisfaction with the experience. It has resulted in assessor and resident satisfaction and has facilitated some change in our teaching practice. This paper will highlight some examples to demonstrate residents' attainment of managerial skills. CONCLUSION: This package enhanced residents' awareness of audit and increased their managerial skills. Services in the health centers, which these residents had tackled, were improved as well.

Journal Article↗
PubMed2003

The role of DPB at anoxic stage in a novel package type BNR process with batch settler.

In this research, package type BNR process was developed to remove nutrients as well as suspended solids from domestic sewage. The effect of HRT reduction of settler on the removal of pollutants as well as the role of DPBs at anoxic reactor were investigated. The proposed package system was composed of sludge denitrification tank, anaerobic, anoxic, oxic, and batch settler. This system could remove nitrogen and phosphorus effectively at low COD/N ratio and also remove SS more effectively than other ordinary BNR system having the conventional settlers. The removal efficiency of total nitrogen (T-N) in optimal condition was about 75.1% under the TCOD/TKN ratio as low as 5.7. The average concentration of the effluent TCOD, ortho-P, NH4+-N, NO3- -N and SS in the package type BNR system of 2 h settler retention time were 15.6. 1.38, 1.4, 10.3 and 3.9 mg/L, respectively. In anoxic state, denitrifying dephosphatation, that is uptaking phosphate by using nitrate-nitrogen as an electron acceptor was observed. The removed NO3- -N concentration by denitrifying dephosphatation was 1.62 mg NO3- -N/mg PO4(3-)-P.

Bacteria, Anaerobic↗
PubMed1970

Segregation and packaging of granule enzymes in eosinophilic leukocytes.

During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.

Acid Phosphatase↗
PubMed2001

Systematic determination of the packaging limit of lentiviral vectors.

Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.

Active Transport, Cell Nucleus↗
PubMed2002

Rapid screening for high-titer retroviral packaging cell lines using an in situ fluorescence assay.

The production of high-titer recombinant retrovirus is a major determinant of the efficiency of target cell transduction. Titer assessment for producer clones that contain vectors encoding proteins that can be detected using fluorescence is typically performed by flow cytometry. However, this method is both costly and labor intensive, severely limiting the number of clones that can be screened for each construct. In this report we describe a rapid, high-throughput screening method for viral quantitation of producer clone supernatant on target cells using a 96-well format. Plates were assayed using a multichannel fluorescent reader to determine the percentage of target cells expressing green (EGFP), cyan (ECFP), yellow (EYFP) or red (DsRed) fluorescent reporter genes, or their combinations. The relative fluorescence counts of target cells incubated with viral supernatant from each packaging cell clone correlated with the level of transduction, and hence, viral titer. Correlation of cell fluorescence between the fluorescent plate reader assay and flow cytometric assessment was high (r(2) = 0.96). Independent detection of different fluorescent reporters enabled multiplex assays to be performed. Simultaneous cell density analysis using alamarBlue fluorescence was proportional to cell number per well (r(2) = 1.0). In situ titer assessment of 66 FLYRD packaging cells encoding the EGFP reporter gene identified clones (>10(7) colony forming units per milliliter [CFU/ml]) that provided titers up to sevenfold over the parent population. The application of this rapid, high-throughput screening method overcomes many limitations imposed by the current flow cytometric screening method. This robust assay maximizes the chance of identifying rare high-titer packaging clones and offers a further opportunity to optimize gene transfer protocols.

Cell Line↗
PubMed1999

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Animals↗
PubMed1997

Retroviral particles produced from a stable human-derived packaging cell line transduce target cells with very high efficiencies.

The goal of this work was to determine whether a stable 293 amphotropic packaging line, which we have designated 293-SPA, is useful for the production of high-titer stable virus by comparison to the murine psiCRIP line. Here, we report our unexpected findings that particles derived from the 293-SPA line transduce target cells (both NIH-3T3 cells and primary melanoma cells) with greatly enhanced efficiencies (at least 10-fold) compared to particles derived from the psiCRIP packaging line. We show that the presence of a transferable inhibitor in the psiCRIP line at least partially accounts for this dramatic difference in transduction efficiency. This work has important implications for improving the efficiency of retrovirus-mediated gene transfer in general as well as in the design of new packaging cell lines.

3T3 Cells↗
PubMed1998

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

Animals↗
PubMed1998

Improvement of retroviral packaging cell lines by introducing the polyomavirus early region.

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

3T3 Cells↗
PubMed2005

Efficient delivery of RNA interference effectors via in vitro-packaged SV40 pseudovirions.

Previously we have shown that in vitro-packaged simian virus 40 (SV40) pseudovirions (IVPs) are an efficient delivery system for supercoiled DNA plasmids of up to 17.7 kb, with or without SV40 sequences. RNA interference (RNAi) is a naturally occurring gene-silencing mechanism mediated by small double-stranded RNA molecules (small interfering RNAs, siRNAs). This study demonstrates the first use of SV40 pseudovirions to deliver into human cells both principal types of RNAi effector molecules: plasmid-expressed short hairpin RNAs (shRNAs) and synthetic siRNAs. We first established the ability of human lymphoblastoid cells to support RNAi, using sequential transduction of .45 cells with packaged plasmid DNA expressing the green fluorescent protein (IVP-GFP), and an shRNA corresponding to the GFP (IVP-shGFP). SV40 mediates DNA transfer of nucleic acid to the cytoplasm, where RNAi-associated cleavage of mRNA principally occurs. Using SV40 pseudovirions, siRNA-mediated RNAi was observed in both .45 cells, after sequential transduction of IVP-GFP and IVP-packaged siRNAs corresponding to GFP (IVP-siGFP), and in HeLa cells stably expressing a GFP transduced with IVP-siGFP. Our findings indicate that SV40 pseudovirions may be a useful addition to the delivery systems currently being used for the transfer of RNAi effector molecules.

Animals↗
PubMed1993

Three peroxisome protein packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis.

We have identified five complementation groups of peroxisome biogenesis (peb) mutants in Saccharomyces cerevisiae by a positive selection procedure. Three of these contained morphologically recognizable peroxisomes, and two appeared to lack the organelle altogether. The packaging of peroxisomal proteins in these mutants has been analyzed with a new gentle cell fractionation procedure. It employs digitonin titration for the selective permeabilization of yeast plasma and intracellular membranes. Proteins were measured by enzymatic assay or by quantitative chemiluminescent immunoblotting. With this gentle fractionation method, it was demonstrated that two mutants are selectively defective in assembling proteins into peroxisomes. Peb1-1 packages catalase and acyl-CoA oxidase within peroxisomes but not thiolase. Peb5-1 packages thiolase and acyl-CoA oxidase within peroxisomes but not catalase. The data suggest that the peroxisome biogenesis machinery contains components that are specific for each of three classes of peroxisomal proteins, represented by catalase, thiolase, and acyl-CoA oxidase. In the two mutants lacking morphologically recognizable peroxisomes, peb2-1 and peb4-1, all three enzymes were mislocalized to the cytosol.

Acetyl-CoA C-Acetyltransferase↗