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PubMed1995

Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.

DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta-galactosidase) triple-fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558, an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is produced intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage T4↗

PubMed1994

Pharmaceutical application of LC-MS. 1--Characterization of a famotidine degradate in a package screening study by LC-APCI MS.

The application of LC-MS to characterize low-level degradates in pharmaceutical dosage formulations is a new and challenging field. In a package screening study, a low-level degradate of famotidine (1, 3-[[[2-[[aminoiminomethyl]-amino]-4-thiazolyl]methyl] thio]-N-(aminosulphonyl)-propanimid-amide, an H2-receptor antagonist, molecular weight: 337) was detected by HPLC in film-coated tablets packaged in child-resistant (CR) foil pouches which were stressed at 40 degrees C/75% relative humidities (RH) for 4 months. LC-atmospheric pressure chemical ionization (APCI) mass spectrometry using positive ion mode yielded a molecular weight of 349 for the degradate, suggesting that it was formed by the addition of one carbon to the famotidine molecule. A detailed analysis of the positive product ion mass spectrum of the protonated degradate ion in a LC-MS-MS study indicated that the carbon was added to the side of N-(aminosulphonyl)-propanimid-amide of famotidine. The structure of the degradate was determined to be 2, which was confirmed by LC-APCI MS and HPLC study of the product formed from the reaction of famotidine with formaldehyde--a one-carbon reagent.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

New, innovative packaging system for surgical sutures and needles.

A new, innovative packaging system for surgical needles and sutures has been developed that meets the special needs of emergency physicians, surgeons, and operating personnel. This packaging system consists of an overwrap, or breather pouch, as well as an innerwrap containing the needle swaged to a surgical suture. The flaps of the overwrap are offset and serrated to facilitate the opening and sterile transfer of the inner packet to the sterile field. The inner packet contains either a plastic labyrinth or craft board that maintains the suture as straight as possible until knot construction. The needle swaged to a suture is parked in foam to protect its sharp cutting edges and point.

Emergency Service, Hospital↗

PubMedDate not supplied

FAMIS: a software package for functional feature extraction from biomedical multidimensional images.

An increased number of image sequences is acquired in all modalities of the biomedical imaging field in order to study displacement or metabolism of a tracer or a contrast agent. It requires effective processing methods to estimate the underlying physiological components. We have developed a software package based on factor analysis algorithms which can adapt to various imaging modalities and its extension to double-indexed image sequences. We describe general characteristics of the software and present the main points of the user-friendly interface. The performances of the package are discussed and the possibilities of the methodology are illustrated using an example in magnetic resonance imaging.

Algorithms↗

PubMedDate not supplied

Role of RNA in bacteriophage phi 29 DNA packaging.

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.

Adenosine Triphosphatases↗

PubMed2004

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes.

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)-BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Animals↗

PubMed2005

Assessment of actinide mass embedded in large concrete waste packages by photon interrogation and photofission.

This paper describes a method based on photofission developed in our laboratory to characterize in depth large waste packages. The method consists in using photons of high-energy (Bremsstrahlung radiation) in order to induce reactions of photofission on the heavy nuclei present in the wastes. The measurement of the delayed neutrons allows quantifying the actinides in the wastes. We present the first results of measurement performed with a concrete mock-up of 870l and two real waste packages.

Actinoid Series Elements↗

PubMed2005

Determination of partition behavior of organic surrogates between paperboard packaging materials and air.

The suitability of recycled paperboard packaging materials for direct food contact applications is a major area of investigation. Chemical contaminants (surrogates) partitioning between recycled paper packaging and foods may affect the safety and health of the consumer. The partition behavior of all possible organic compounds between cardboards and individual foodstuffs is difficult and too time consuming for being fully investigated. Therefore it may be more efficient to determine these partition coefficients indirectly through experimental determination of the partitioning behavior between cardboard samples and air. In this work, the behavior of organic pollutants present in a set of two paper and board samples intended to be in contact with foods was studied. Adsorption isotherms have been plotted and partition coefficients between paper and air have been calculated as a basis for the estimation of their migration potential into food. Values of partition coefficients (Kpaper/air) from 47 to 1207 were obtained at different temperatures. For the less volatile surrogates such as dibutyl phthalate and methyl stearate higher Kpaper/air values were obtained. The adsorption curves showed that the more volatile substances are partitioning mainly in air phase and increasing the temperature from 70 to 100 degrees C their concentrations in air (Cair) have almost doubled. The analysis of surrogates was performed with a method based on solvent extraction and gas chromatographic-flame ionization detection (GC-FID) quantification.

Air↗

PubMed2006

A proposed model of fat packaging by exchangeable lipid droplet proteins.

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Adipose Tissue↗

PubMed2005

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging.

OBJECTIVE: Our goal was to test the hypothesis that spermatozoal chromatin packaging changes with age and that aging affects the susceptibility of spermatozoal DNA to oxidative damage. DESIGN: Laboratory study. SETTING: Academic facility. PATIENT(S): Young (4 months) and old (21 months) Brown Norway rats. INTERVENTION(S): Spermatozoa were collected from the cauda epididymidis and were incubated in saline or H2O2. MAIN OUTCOME MEASUREMENT(S): Thiols levels, chromatin condensation, DNA susceptibility to acid-induced DNA denaturation, and DNA damage were evaluated using monobromobimane, chromomycin A3 (CMA3), acridine orange, and polymerase chain reaction, respectively. RESULT(S): Spermatozoa from old rats had 25% fewer disulfides but similar levels of free thiols as compared with young. The CMA3 staining was decreased by 13% with age. Levels of chromatin denaturation and DNA damage were similar in control groups. After exposure to oxidant, free thiols became oxidized by about 20% irrespective of age, but CMA3 staining changed little. The acridine orange assay, however, showed a trend for greater chromatin denaturation in spermatozoa from old rats after oxidant treatment. Furthermore, the DNA from spermatozoa of old rats was significantly more susceptible to developing DNA breaks and modification after oxidative challenge. CONCLUSION(S): Spermatozoal chromatin packaging changes with aging and vulnerability to oxidative damage increases.

Aging↗

PubMed2006

The potential of retroviral vectors to cotransfer human endogenous retroviruses (HERVs) from human packaging cell lines.

Using a versatile and highly sensitive retroviral microarray, we have investigated particle preparations from three different human packaging cell lines harboring retroviral vector systems based on human immunodeficiency virus (HIV) and murine leukemia virus (MLV). 293Rev/Gag/Pol(i) cells inducibly express high titers of HIV-derived particles for packaging of HIV vectors. The Phoenix-GP and the Anjou 65 cell lines constitutively express MLV vector particles. We compared the transcription profiles of human endogenous retroviruses (HERVs) in all cell lines with the HERV sequences present in the particles. In addition, the influence of the transfected vector plasmid on the copackaging of HERVs was investigated. All particle preparations showed a defined pattern of endogenous retroviral sequences that differed from the cellular HERV expression pattern. HERV transcripts were observed in the particle preparations independent of whether a vector construct was coexpressed or not. Furthermore, our results suggest that particle preparations are frequently contaminated by cellular vesicles (exosomes) containing cellular RNAs including HERV transcripts.

Animals↗

PubMed2004

Criteria and procedures for determining benefit packages in health care. A comparative perspective.

The legitimacy of procedures and criteria for determining benefit packages depends crucially on the representation of stakeholders in decision-making bodies, the transparency of procedures and the consistency of benefit decisions. Moreover, the assessment of the costs of healthcare services and its application as a decision criterion can be an important policy instrument in order to increase the overall efficiency of healthcare systems. Our analysis of procedures and criteria for determining benefit packages in England, Germany and Switzerland established potential for developing more legitimate procedures and criteria for benefits decisions. In Germany, representation of stakeholders and transparency of procedures can be improved. Consistency of decision-making is hindered by the veto positions of selected stakeholders. Moreover, benefit decisions are made for different healthcare sectors separately. In Switzerland, transparency of procedures is virtually non-existent at the moment. Thus, it is impossible to assess the consistency of decision-making. Only in England the costs of healthcare services influence the decision to include or exclude them.

Cost Sharing↗

PubMed2006

Structure of a hexameric RNA packaging motor in a viral polymerase complex.

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.

Cryoelectron Microscopy↗

PubMed2006

From leading role to the backstage: mesenchymal stem cells as packaging cell lines for in situ production of viral vectors.

Gene therapy is based on the genetic manipulation of target cells. The genetic information required to genetically engineer these cells can be delivered through non-viral or viral vectors that present different biologic properties. The production of viral vectors for gene therapy depends on the nature of the cells transfected with plasmids containing the genetic information for recombinant viral assemblage. These so-called packaging cell lines (PCL) can be injected into the target organ, for the in situ transduction of target cells. There have been recent reports about the capacity of mesenchymal stem cells (MSCs) to target tumor cells. Different research groups, including our own, have isolated these MSCs, but they have not yet been studied as potential PCL to produce viral vectors. We propose here that a MSC packaging cell line could be employed for in situ gene therapy of solid tumors. The tropism of MSCs for tumor cells may render this PCL more efficient in that microenvironment, producing viral vectors for longer periods of time, shifting MSCs from target cell to the backstage level of viral gene therapy.

Adenoviridae↗

PubMed2005

simBio: a Java package for the development of detailed cell models.

Quantitative dynamic computer models, which integrate a variety of molecular functions into a cell model, provide a powerful tool to create and test working hypotheses. We have developed a new modeling tool, the simBio package (freely available from ), which can be used for constructing cell models, such as cardiac cells (the Kyoto model from Matsuoka et al., 2003, 2004 a, b, the LRd model from Faber and Rudy, 2000, and the Noble 98 model from Noble et al., 1998), epithelial cells (Strieter et al., 1990) and pancreatic beta cells (Magnus and Keizer, 1998). The simBio package is written in Java, uses XML and can solve ordinary differential equations. In an attempt to mimic biological functional structures, a cell model is, in simBio, composed of independent functional modules called Reactors, such as ion channels and the sarcoplasmic reticulum, and dynamic variables called Nodes, such as ion concentrations. The interactions between Reactors and Nodes are described by the graph theory and the resulting graph represents a blueprint of an intricate cellular system. Reactors are prepared in a hierarchical order, in analogy to the biological classification. Each Reactor can be composed or improved independently, and can easily be reused for different models. This way of building models, through the combination of various modules, is enabled through the use of object-oriented programming concepts. Thus, simBio is a straightforward system for the creation of a variety of cell models on a common database of functional modules.

Algorithms↗

PubMed2006

Evidence for specific packaging of the influenza A virus genome from conditionally defective virus particles lacking a polymerase gene.

The incorporation of the eight gene segments of influenza A virus in virions, either through a random or a specific mechanism, has been under debate for many years. Using reverse genetics techniques and trans-complementation of viral proteins, we showed that the production of viruses containing seven gene segments is very inefficient, which can be overcome by providing the 8th gene segment in a non-functional form. We conclude that sequences in the 5' and 3' ends of the polymerase gene segments contain packaging signals. Our methods will facilitate the further mapping of the packaging signals of influenza A virus.

DNA-Directed RNA Polymerases↗

PubMed2004

Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor.

In cells infected by HIV-1 mutants lacking a functional Vif protein, APOBEC3G is specifically packaged into progeny virions and then interferes with the process of virus infection. Here, we show that incorporation of APOBEC3G into HIV-1 virions is mediated by the specific interaction of APOBEC3G with the carboxy-terminal nucleocapsid/p6 domain of the Gag polyprotein precursor. As a result, HIV-1 virus-like particles that lack the nucleocapsid domain fail to package APOBEC3G. Surprisingly, RNA was also found to be essential for formation of the nucleocapsid--APOBEC3G complex in vitro, thus raising the possibility that RNA may form a bridge between these two proteins.

APOBEC-3G Deaminase↗

PubMed2005

The R362A mutation at the C-terminus of CA inhibits packaging of human immunodeficiency virus type 1 RNA.

The capsid (CA) sequence of human immunodeficiency virus type 1 (HIV-1) Gag protein consists of two independently folded domains named the N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by a flexible linker. Most of the CTD sequence adopts rigid structure except for the last 11 amino acids (positions 354 to 364) that are disordered even in the context of the downstream SP1 and nucleocapsid (NC) sequence. Although disordered, this short peptide region plays a crucial role in HIV-1 replication. In this study, we identified three second-site mutations within Gag named A238T, G358S, and N373K that rescued a deleterious mutation R362A located at the C-terminus of CA. A238T is located within the NTD of CA, G358S and N373K are positioned proximal to R362A. One of the mechanisms underlying this compensation event is correction of reduced packaging of viral RNA into the R362A mutated viruses, as shown by the results of RNase protection assays, native Northern blots experiments as well as filter-binding assays. These data suggest that one potential function for the C-terminal disordered sequence of CA in HIV-1 replication is to regulate HIV-1 RNA packaging.

Amino Acid Sequence↗