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At least 379 records · Page 21Linked to original sources
PubMed1992

A phage T4 in vitro packaging system for cloning long DNA molecules.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.

Bacteriophage lambda↗
PubMed2004

The tRNALys packaging complex in HIV-1.

The major human tRNALys isoacceptors, tRNALys1,2 and tRNALys3, are selectively packaged into HIV-1 during assembly, where tRNALys3 acts as the primer for initiating reverse transcription. In this report, we shall review the evidence that supports a model for the formation of a tRNALys packaging complex, whose components include the precursor proteins Gag and Gag-Pol, viral genomic RNA, tRNALys, and lysyl-tRNA synthetase (LysRS). In the model proposed, the tRNALys packaging complex is formed when a Gag/Gag-Pol/viral RNA complex interacts with a tRNALys/LysRS complex, with Gag interacting with LysRS, and Gag-Pol interacting with tRNALys. The incorporation of Gag-Pol into HIV-1 requires its interaction with Gag multimers whose polymerization is promoted by RNA. Reverse transcriptase sequences within Gag-Pol also bind to tRNALys, and this binding is required for tRNALys packaging into viruses. LysRS, the enzyme that aminoacylates tRNALys, is also incorporated into HIV-1, and this protein is a strong candidate for being the signal that specifically targets tRNALys for viral incorporation. Newly-synthesized LysRS is a main source of viral LysRS, and its incorporation into viruses occurs via its interaction with Gag and independently of tRNALys packaging. While tRNALys incorporation into viruses depends upon its interaction with LysRS, tRNALys aminoacylation is not a requirement for viral packaging.

Aminoacylation↗
PubMed2005

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus.

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.

Amino Acid Sequence↗
PubMed2005

Enhanced long-term expression from helper virus-free HSV-1 vectors packaged in the presence of deletions in genes that modulate the function of VP16, U L 46 and U L 47.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off recombinant gene expression from HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus paradoxically, recombinant gene expression remains short-term in the absence of almost all (approximately 99%) of the HSV-1 genome. To resolve this paradox, we hypothesize that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. In an earlier study, we examined the effects on recombinant gene expression of five different proteins that affect the HSV-1 virion. We found that vectors packaged in the presence of mutated vhs or U S 11 exhibited minimal changes in gene expression, vectors packaged in the presence of a mutated U S 3 supported improved gene transfer (numbers of cells at 4 days), and vectors packaged in the presence of mutated U L 13 or VP16 supported improved long-term expression. The capability of the VP16 transcriptional complex to reduce gene expression deserves additional study because VP16 is a powerful enhancer that interacts with a number of cellular and viral proteins. In particular, U L 46 and U L 47 are known to modulate the effects of VP16 on immediate early promoters. In this study, we examined expression from a HSV-1 vector that contains a neuronal-specific promoter and was packaged in the presence of deletions in U L 46, or U L 47, or both U L 46 and U L 47. In the rat striatum, each of these vector stocks supported both improved gene transfer (numbers of cells at 4 days) and improved long-term expression (2 months). Vectors packaged in the presence of a deletion in both U L 46 and U L 47 supported larger improvements in gene expression compared to vectors packaged in the presence of deletions in either gene alone. The implications of these results for strategies to improve long-term expression are discussed.

Animals↗
PubMed2003

A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.

Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+ cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+ cells. Viral particles from this cell line were concentrated up to 100-fold (up to 10(7) viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a Neo(R)-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. We also show transfer of a human beta-globin gene into progenitor cells from CD34+ cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.

ADP-ribosyl Cyclase↗
PubMed2003

Selective transport and packaging of the major yolk protein in the sea urchin.

The major yolk protein of sea urchins is an iron-binding, transferrin-like molecule that is made in the adult gut. Its final destination though is the developing oocytes that are embedded in somatic accessory cells and encompassed by two epithelial layers of the ovary. In this study, we address the dynamics of yolk transport, endocytosis, and packaging during the vitellogenic phase of oogenesis in the sea urchin by use of fluorescently labeled major yolk protein (MYP). Incorporation of MYP into the accessory cells of the ovary and its packaging into yolk platelets of developing oocytes is visualized in isolated oocytes, ovary explants, and in whole animals. When MYP is introduced into the coelom of adult females, it is first accumulated by the somatic cells of the ovarian capsule and is then transported to the oocytes and packaged into yolk platelets. This phenomenon is specific for MYP and accurately reflects the endogenous MYP packaging. We find that oocytes cultured in isolation are endocytically active and capable of selectively packaging MYP into yolk platelets. Furthermore, oocytes that packaged exogenous MYP are capable of in vitro maturation, fertilization, and early development, enabling an in vivo documentation of MYP utilization and yolk platelet dynamics. These results demonstrate that the endocytic uptake of yolk proteins in sea urchins does not require a signal from their surrounding epithelial cells and can occur autonomous of the ovary. In addition, these results demonstrate that the entire population of yolk platelets is competent to receive new yolk protein input, suggesting that they are all made simultaneously during oogenesis.

Animals↗
PubMed1998

Possible objectives and resulting entitlements of essential health care packages.

The notion of a defined 'core package of essential health care services' has appeared in many different health reform proposals in the 1990s. This paper attempts to explore the possible objectives of the 'core package' component of health care reform. Two board applications are apparent: the use of essential packages to ration scarce public funds and the incorporation of a minimum benefit package into 'managed competition' type reforms, where they constitute a mandated minimum level of private insurance cover. Eight possible objectives for an essential benefit package are described: To protect against catastrophic illness events; to ensure social risk pooling; to improve allocative efficiency in the health system; to eliminate 'high burden of disease' conditions; to improve equity of access to services; to combat cost-escalation; to encourage competition between insurers; and to facilitate public participation and transparency in decision making. Closer examination of objectives reveals that they often conflict, which suggests that a clear understanding of the purpose of reform is essential before it is worthwhile devoting energy to the development of essential benefit packages. It is argued that two main clusters of objectives emerge from the eight described, representing Rawlsian (risk avoidance) and utilitarian (efficiency improvement) social welfare philosophies, respectively. Practical experience suggests that priority setting exercises have been unsuccessful in meeting efficiency objectives, but that they may well be quite useful in fulfilling risk-pooling aims.

Cost-Benefit Analysis↗
PubMedDate not supplied

3DVIEWNIX-AVS: a software package for the separate visualization of arteries and veins in CE-MRA images.

Our earlier study developed a computerized method, based on fuzzy connected object delineation principles and algorithms, for artery and vein separation in contrast enhanced Magnetic Resonance Angiography (CE-MRA) images. This paper reports its current development-a software package-for routine clinical use. The software package, termed 3DVIEWNIX-AVS, consists of the following major operational parts: (1) converting data from DICOM3 to 3DVIEWNIX format, (2) previewing slices and creating VOI and MIP Shell, (3) segmenting vessel, (4) separating artery and vein, (5) shell rendering vascular structures and creating animations. This package has been applied to EPIX Medical Inc's CE-MRA data (AngioMark MS-325). One hundred and thirty-five original CE-MRA data sets (of 52 patients) from 6 hospitals have been processed. In all case studies, unified parameter settings produce correct artery-vein separation. The current package is running on a Pentium PC under Linux and the total computation time per study is about 3 min. The strengths of this software package are (1) minimal user interaction, (2) minimal anatomic knowledge requirements on human vascular system, (3) clinically required speed, (4) free entry to any operational stages, (5) reproducible, reliable, high quality of results, and (6) cost effective computer implementation. To date, it seems to be the only software package (using an image processing approach) available for artery and vein separation of the human vascular system for routine use in a clinical setting.

Algorithms↗
PubMed2003

Long-term storage of small surgical instruments in autoclaved packages.

BACKGROUND: In most operating theatres, unused sterile instruments must be re-sterilized according to preset protocols. Protocols differ among institutions and are not based on strong scientific evidence. OBJECTIVE: To determine and compare the duration of sterility of small instruments packaged in double-layered linen versus plastic-paper envelopes after autoclaving. MATERIALS AND METHODS: Two groups of orthopaedic screws were simultaneously sterilized by autoclaving. In Group 1, each screw was packaged in a double-wrapped linen pack. The screws in Group 2 were individually packaged in an inner wrap of paper and an outer plastic-paper envelope that is commercially available. Unwrapped screws in Group 3 served as controls. During the first 48 weeks, five packages were randomly taken from each group, and from 48 weeks to 96 weeks, 20 packages were taken at random and sent for microbial culture. Five screws from Group 3 were also randomly picked with each sample. RESULTS: Up to 96 weeks, no organisms were cultured from any sample from Groups 1 and 2. Almost all samples from Group 3 grew several species of bacteria. CONCLUSION: For small metal instruments, autoclaved packages in double-wrapped linen or double-wrapped plastic-paper combinations can be stored safely for at least 96 weeks.

Colony Count, Microbial↗
PubMed2003

Packaging of an AAV vector encoding human acid alpha-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector.

We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid alpha-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10(10) AAV vector particles. These modifications resulted in approximately 30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid alpha-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.

Adenoviridae↗
PubMed1999

Packaging and replication regulation revealed by chimeric genome segments of double-stranded RNA bacteriophage phi6.

Bacteriophage phi6 has a double-stranded RNA genome composed of three linear segments, L, M, and S. The innermost particle in the virion of phi6, like in the other dsRNA viruses, is an RNA-dependent RNA polymerase complex, which carries out all the functions needed for the replication of the viral genome. Empty polymerase complexes can package the single-stranded copies of the viral genome segments, replicate the packaged segments into double-stranded form (minus strand synthesis), and then produce new plus strands (transcripts) from the double-stranded RNA templates. The three viral genomic segments contain unique packaging signals at their 5' ends, and minus strand synthesis initiation is dependent on the sequence at the 3' end. Here we have constructed chimeric segments that have the packaging signal from one segment and the minus strand synthesis initiation signal from another segment. Using purified recombinant polymerase complexes and single-stranded/chimeric and original RNA segments, we have analyzed the packaging and replication regulation operating in in vitro conditions. We show that the 5' end of the L genome segment in single-stranded form is needed to switch from the packaging to the minus strand synthesis and the same sequence is required in double-stranded form to switch on plus strand synthesis. In addition we have constructed deletions to the M segment to analyze the possible regulatory role of the internal noncoding area of this segment.

Bacteriophage phi 6↗
PubMed2006

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells.

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.

B-Lymphocytes↗
PubMed2000

Contraception: a new practical learning package.

BACKGROUND: A new self-directed learning package was developed to assist medical students learn the counselling and practical skills to enable them to communicate with men and women about contraception and related matters. OBJECTIVES: This paper describes the package and the way it was facilitated, the students' ratings of the package and their feedback about the session on the first time it was presented at four teaching hospitals. RESULTS: The students rated the contraception package as average. The reasons given were: no introduction to the contraception session, lack of a trained person to conduct and facilitate the contraception learning session, poor organization at one of the hospitals and too little emphasis on self-assessment. The assumption that the students had a basic hormonal knowledge prior to the contraception sessions was incorrect. DISCUSSION: The results suggest the contraception learning package needs a person with contraceptive knowledge, patient-doctor skills and experience with self-directed learning to be present throughout the 3-hour session and for tools to be available that emphasize self-assessment during the session. OUTCOME: The modifications to be made to the learning package include pairing male and female students, a reduction in duration of the learning stations, an additional learning station relating to hormonal contraception, and inclusion of pregnancy and ovulation testing. These modifications were suggested by the participating students.

Adult↗
PubMed2003

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Animals↗
PubMed1992

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.

Animals↗
PubMed1997

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6.

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Bacteriophages↗
PubMedDate not supplied

Legislation, control and research in the Nordic countries on plastics for packaging food.

The present legislation in the Nordic countries for food contact materials is expressed in general terms and contains few detailed requirements. At present Finland is implementing the EEC legislation, Sweden and Norway will probably do so shortly and Denmark has been a member of the EEC since 1973. Current food legislation in Sweden only covers materials or articles intended to come into contact with foodstuffs during processing or packaging in the food industry or by retailers. It does not apply to food packaging materials purchased for use at home or to household utensils. Upon request, the Toxicology Division at the Swedish National Food Administration (NFA) carries out evaluations of materials intended to come into contact with food. In addition, a voluntary organization--Normpack--is currently operating in Sweden. Normpack consists of manufacturers, dealers and users of food packaging materials, who have agreed to abide by certain common standards. In Norway, the Packaging Convention (Emballasjekonvensjonen--on safety of food packaging material from the health point view) serves a similar purpose. Research in this field is conducted at the National Food Agency of Denmark, The Danish Packaging and Transportation Research Institute (ETi) of the Danish Technological Institute (DTI), the Food Research Laboratory at the Technical Research Centre of Finland, MATFORSK, Norconserv and Statoil in Norway and the NFA, PackForsk and the Swedish Institute for Food Research (SIK) in Sweden. Previous studies have concerned plasticizers in PVC (polyvinyl chloride) cling film, overall migration studies on cling film, specific migration of vinyl chloride, styrene and acrylonitrile and off-flavours.(ABSTRACT TRUNCATED AT 250 WORDS)

European Union↗
PubMedDate not supplied

United States of America and European regulation of food packaging: finding common ground to reach a common goal.

There are pronounced differences between the USA approach to the regulation of food packaging and the system being adopted in the European Economic Community (EEC). These differences have significant implications for the efficient regulation of packaging materials and for achieving the common goal of regulatory harmonization. The United States effects preclearance of packaging materials by generic regulation, a modified 'positive list' system with certain jurisdictional exclusions. Distinguishing characteristics of the USA system include exemptions for materials that are 'prior sanctioned', 'generally-recognized-as-safe' (GRAS) or 'not reasonably expected to become a component of food'. The USA also embraces the application of the concept of Estimated Dietary Intake and use limitations to take into account likely exposure and, thereby, delimit the requirements for toxicological data. The EEC, on the other hand, is moving towards adoption of a strict positive list system under which no substance may be used in making a package or packaging material unless it is on the positive list on the basis of a toxicological conclusion as to the general safety of the substance. This paper examines the logical and philosophical underpinnings of both systems and the potential for common sense application of de minimis, or regulatory threshold principles, and worldwide use of the Estimated Dietary Intake concept, to help bring about a measure of harmonization consistent with the safe and efficient regulation of food packaging materials.

European Union↗