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At least 667 records · Page 37Linked to original sources
PubMed1998

Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors.

Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.

Animals↗
PubMed2006

The importance of written information packages in support of case-finding within families at risk for inherited high cholesterol.

Inherited High Cholesterol is treatable, but highly underdiagnosed. To detect undiagnosed blood relatives at a presymptomatic stage, in the Netherlands written information packages are available to facilitate family communication. To investigate the role of those packages in the detection of carriers, we conducted a qualitative evaluation (plus-minus method combined with semistructured interviews with index patients and relatives). Our data suggest that interviewees approved the family approach for finding carriers, although reluctantly. The packages aided family disclosure by reducing hesitation. However, index patients only informed first-degree relatives and generally communicated the risk only once. This may be due to the cultural context and a limited understanding of genetics. For relatives the packages served as a cue to action and as a legitimation to gain access to a diagnostic cholesterol test. Despite the value of these written materials, they should not be used as the only communication between index patient and relatives.

Adult↗
PubMed1985

A computer program package for relative survival analysis.

A computer program package has been constructed for use in patient survival analyses for chronic diseases based on aggregated data. The central concept of the analyses--the relative survival rate--is the ratio of the observed survival rate of the patients to the survival rate expected in a group in the general population similar to the group of patients at the beginning of the follow-up (interval), with respect to age, sex and calendar time. This quantity is used to measure patient survival adjusted for the effect of mortality attributable to the competing risks of death without employing information on causes of death of individual patients. The package contains three alternative methods of estimating the relative survival rates, two different ways of estimating the expectation of life for the patients, and five methods of testing the relative survival patterns using information on the whole follow-up period. Conventional survival and competing risk analysis can also be performed with the package. It is hoped that the package will facilitate standardization of statistical methodology and terminology in long-term survival studies for chronic diseases.

Aged↗
PubMed1993

A hardware-independent data acquisition package incorporating a simulation driver and simulation description language.

Device drivers for commercial data acquisition boards (DASH-16, DT2801) and a device driver for a virtual data acquisition board have been developed and incorporated in a single package for a multiple station anesthesia research laboratory. The package provides a compiler for a simulation description language which is used to program the virtual board. The package, written in C for the IBM PC, was designed to be easily extended with additional drivers. Applications using the package can control all supported devices without the programmer having to learn the details of the hardware.

Computer Simulation↗
PubMed1993

Elucidation of a conserved RNA stem-loop structure in the packaging signal of human immunodeficiency virus type 1.

A region of human immunodeficiency virus type 1 (HIV-1) genomic RNA known to interact with the viral nucleocapsid protein is one of the crucial components for retroviral genomic RNA packaging. We have investigated the secondary structure for this RNA packaging signal of HIV-1 using RNA mapping techniques and extensive computer analyses. Our results suggest that the RNA sequence containing the packaging signal conforms to a stable stem-loop structure and that a portion of this structure is conserved among twenty independent HIV-1 isolates. The heterogeneity of the RNA packaging sequence was not correlated with the amino acid sequence variability of the viral nucleocapsid protein. These findings have critical implication in understanding the viral maturation process.

Amino Acid Sequence↗
PubMedDate not supplied

Development of image analysis demonstration packages with MENU: educational aspects.

In this paper a tool, MENU, is presented, with which demonstration packages can be easily constructed. The teacher designs the set-up of the package by editing a demonstration specification file, containing both commands to MENU to display frames to the end-user or to execute tasks and the text of the frames. The text contains explanations for the end-user together with the options he can choose. MENU takes care that the corresponding actions are executed. Two image analysis packages, one about CT and one about gated cardiac bloodpool scintigraphy, are presented as examples of the use of MENU. It is concluded, that with MENU (existing) programs can be modeled into packages very easily and efficiently. MENU proves to be a tool that is worthwhile for educational purposes.

Computers↗
PubMed1983

Tests of spool models for DNA packaging in phage lambda.

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗
PubMed1987

Initiation events in in-vitro packaging of bacteriophage phi 29 DNA-gp3.

Initiation events in the packaging of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were studied in a completely defined in-vitro system that included purified proheads, DNA-gp3 and the DNA packaging protein gp16. In the sequential interactions, gp16 first bound to, and was modified by, the prohead. The prohead-gp16 complex then bound to DNA-gp3, resulting in a second modification of gp16 that permitted binding of ATP. DNA-gp3 aggregates were produced, and the hydrolysis of ATP accompanied DNA-gp3 packaging. Binding and hydrolysis of ATP by gp16 was both prohead- and DNA-gp3-dependent. Interruption of packaging by DNase I addition revealed filled heads but few particles containing partial lengths of DNA, suggesting that following a rate-limiting initiation, the translocation of DNA-gp3 into the prohead was much faster in the defined in-vitro system than in extracts.

Adenosine Triphosphate↗
PubMed1987

Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29.

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.

Adenosine Triphosphatases↗
PubMed1985

In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro.

In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length.

DNA Transposable Elements↗
PubMed1985

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B.

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Capsid↗
PubMed1987

A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage lambda FI gene product.

A new in vitro bacteriophage lambda DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells. This assay is used to demonstrate that the lambda FI gene product (gpFI) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts. A 100- to 200-fold decrease in packaging is seen when gpFI is omitted. gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex.

Bacteriophage lambda↗
PubMed1991

sRNA of phage phi 29 of Bacillus subtilis mediates DNA packaging of phi 29 proheads assembled in Escherichia coli.

The structural genes of the prohead of phage phi 29 of Bacillus subtilis and a small phi 29 RNA (sRNA) were cloned and expressed in Escherichia coli individually or in combination to study the role of the sRNA in prohead assembly and the mechanism of prohead morphogenesis. The genes coding for the proteins of the scaffold (gp7), the capsid (gp8), the portal vertex (gp10), and the dispensable head fiber (gp8.5) were expressed in E. coli and the gene products were assembled, with and without the presence of the sRNA, into uniform and prolate particles that resembled the typical native phi 29 prohead. No differences in particle size and shape were found between the particles of 7-8-8.5-10 (scaffold-capsid-fiber-portal vertex) and 7-8-8.5-10-RNA (scaffold-capsid-fiber-portal vertex-RNA), suggesting that the phi 29 sRNA was not required for phi 29 prohead assembly. The 7-8-8.5-10 particles produced in E. coli in the absence of phi 29 sRNA were fully competent to package phi 29 DNA in the defined in vitro DNA packaging system by the addition of purified sRNA. Moreover, these DNA-filled heads were assembled into infectious virions in extracts. Without the addition of the sRNA, the 7-8-8.5-10 particles were incompetent while the 7-8-8.5-10-RNA particles were competent in DNA packaging. Bacterial sRNA present in E. coli cannot substitute for the phi 29 sRNA. The assembly of prohead particles in E. coli indicated that host factors unique to B. subtilis were not required. The evidence that the phi 29 sRNA was not required for phi 29 prohead assembly and was not a fixed structural component of the phi 29 prohead favors the conclusion that the phi 29 sRNA is a specific enzyme or morphogenetic factor in DNA packaging.

Bacillus subtilis↗
PubMed1994

Comparative study of two in vitro models (L-929 fibroblasts and Tetrahymena pyriformis GL) for the cytotoxicological evaluation of packaged water.

The preservation of 234 samples of water packaged in polychloride vinyl (PVC), polyethylene terephthalate (PET) and glass-bottles was concurrently evaluated using both classical chemical analysis oriented to finding organic and inorganic substances which could have been leached by the packaging and in vitro toxicological bioassays using two models: the L-929 established cell line of fibroblasts and the ciliated protozoa Tetrahymena pyriformis GL. Although no significant abnormality was detected with the chemical analysis, cytotoxicity was detected with both models in some samples of water bottled for > 18 months, for either PVC, PET or glass-packaging. However, statistical analysis did not allow a mathematical relationship between the cytotoxic effects, the length of storage and the packaging to be demonstrated.

Animals↗
PubMed1990

The storage life of chicken carcasses packaged under carbon dioxide.

Broiler chicken carcasses were packaged under vacuum in film of low oxygen transmission rate, or under CO2 in gas-impermeable aluminum foil laminate. The packaged carcasses were stored at +3 or -1.5 degrees C. The initial flora was dominated by enterobacteria. Vacuum-packaged carcasses developed microbial populations in which enterobacteria continued to predominate, and were spoiled by persistent putrid odours after 2 weeks storage at 3 degrees C or 3 weeks storage at -1.5 degrees C. Growth of enterobacteria was inhibited on carcasses packaged under CO2, the microflora that developed being dominated by lactobacilli. However, slow growth of the enterobacteria eventually resumed, and putrid spoilage was apparent after 7 weeks storage at 3 degrees C or 14 weeks storage at -1.5 degrees C.

Animals↗
PubMed1994

Subgenomic S1 segments are packaged by avian reovirus defective interfering particles having an S1 segment deletion.

Two subgenomic segments derived from the S1 genome segment (SGS1-1 and SGS1-2) were identified in avian reovirus defective interfering (DI) particle preparations having an S1 segment deletion. Both SGS1-1 and SGS1-2 were composed of double-stranded RNA (dsRNA) with an estimated size of 400 and 380 bp, respectively. Their segment of origin was identified as the S1 by hybridization analysis. The subgenomic segments were associated with the virus fraction following CsCl density gradient centrifugation, indicating that they are packaged. The subgenomic segments were also shown to be replicated. Therefore, sequence(s) required for replication and packaging are retained. The relative amounts of subgenomic segments were shown to be inversely proportional to that of the S1 segment. The presence of subgenomic segments and concurrent reduction in the relative amount of the S1 segment were found to be directly associated with the decrease in infectious titers. These results suggest that subgenomic segments are responsible for induction of interference by specifically competing with the S1 segment during replication and/or packaging. The competitive relationship between the subgenomic segments and the S1 segment implies that segment-specific sequence(s) or factor(s) are involved in the replication and/or packaging of each individual genome segment.

Animals↗
PubMed1989

QUAD: a computer package for the analysis of QUantal Assay Data.

The computer package QUAD has been developed at the University of Kent, U.K. It is menu driven and written in Advanced BASIC. It runs on IBM PC compatible machines equipped with a suitable graphics facility such as CGA or simulated CGA. QUAD is available on a floppy disk, for a small handling charge. QUAD has four main functions: it performs a logit analysis of quantal assay data; it provides a flexible way of analysing the data, allowing dose transformations and providing alternative confidence intervals for EDp values; it produces a range of diagnostics for assessing the fit of models to data; it provides and fits two families of extended models, each containing the logit as a special case. The package makes use of the latest statistical research, and fitted models are displayed by means of the good graphics facilities available on microcomputers. This document describes the facilities available in detail, and provides and discusses, illustrations of the package at work. QUAD has been designed as a pilot package. Further additions and developments are planned and described later.

Animals↗
PubMed1989

Morpheus: a conformation-activity relationships and receptor modeling package.

Our molecular modeling software package, MORPHEUS, allows the study of the interactions between biologically active molecules and their receptors. The package is capable of exploring the multidimensional conformational space accessible to each molecule of the data set under study. By specifying distance constraints or hypothetical receptor binding points, the package is able to filter the biologically accessible conformations of each active compound and deduce a three-dimensional model of the binding sites consistent with the properties of the agonists (or antagonists) under scrutiny. The electrostatic potentials in the environment of a putative binding site can also be investigated using the MORPHEUS package. The molecular modeling module CRYS-X, which is written in FORTRAN 77 for IBM PC machines, is capable of building, displaying and manipulating molecules.

Computer Graphics↗