Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Cholesterol photosensitized oxidation in food and biological systems

Lipid oxidation is one of the main chemical degradations occurring in biological systems and leads to the formation of compounds that are related to aging and various chronic and degenerative diseases. The extent of oxidation will depend on the presence of antioxidants/pro-oxidants, the unsaturation degree of fatty acids, and environmental conditions. Lipid oxidation can also affect other molecules that have double bonds in their chemical structures, such as cholesterol. Cholesterol oxidation products (COPs) have been studied in depth, because of their negative and controversial biological effects. The formation of COPs can be particularly favored in the presence of light and photosensitizers, since they generate excited singlet oxygen that rapidly reacts with the double bond by a non radical mechanism and without any induction period. The present review intends to provide an overall and critical picture of cholesterol photosensitized oxidation in food and biological systems, and its possible impact on human health and well-being.     

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.     

Specific short hairpin RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus

To clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles.     

药品包装材料与b型流感嗜血杆菌结合疫苗相容性研究

目的考察不同组合的内包装材料对Hib结合疫苗的影响,选出最适合的包装材料。方法对所选药品包装材料按照生产厂家的不同来搭配,共得到12种组合,根据相容性试验设计,将样品置于25±2℃环境下,分别在1、2、3、6个月时取样,进行pH值、内毒素、不溶性微粒3个项目的检测。结果对pH值、内毒素含量、不溶性微粒检测数据进行分析,选出了对Hib结合疫苗影响最小的包装材料组合C+Y组。结论一个适宜的包装内环境对产品的稳定性十分重要。     

Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

The p32K Structural Protein of the Atadenovirus Might Have Bacterial Relatives

Abstract The primary structure of a novel adenoviral protein referred to as p32K and found exclusively in members of the proposed new genus Atadenovirus was analyzed. The p32K gene sequence was determined from two bovine and one snake adenovirus types. Altogether five different p32K sequences were examined, two of them were obtained from the Gene Bank. The C-terminal part of the protein is conserved and shares similarity with certain bacterial small acid soluble proteins (SASPs). The sequence similarity seems coupled with functional relatedness, i.e. both protein groups are found in structures where the genome of the “dormant” organism is packaged in tight nucleoprotein complexes. In these complexes the DNA is protected against harmful environmental effects until the new reproductive cycle is started with specific protease cleavage of the packaging proteins. Although there is no experimental clue about the role of the p32K proteins, we hypothesize phylogenetic relationship between the two protein groups based on the sequence similarity and the supposed functional similarity. The alignments of these protein groups shows that the conserved part of the p32Ks probably is the result of the duplication of a shorter sequence similar to the SASPs of the Bacilli.     

Properties of baked foams based on cassava starch, sugarcane bagasse fibers and montmorillonite

The objectives of this work were to develop biodegradable trays from cassava starch, sugarcane fibers and Na-montmorillonite (Na-MMT) using a baking process and to study the effects of these components on the microstructure and physicochemical and mechanical properties of the trays. All formulations resulted in well-shaped trays with densities between 0.1941 and 0.2966 g/cm³. The addition of fibers and Na-MMT resulted in less dense and less rigid trays. As observed in the water sorption isotherms, the increase in the equilibrium moisture content was more pronounced when the samples were stored at RH (relative humidity) above 75%. The foams had high water absorption capacities (>50%) when immersed in water (1 min). The studied processing conditions resulted in good nanoclay dispersion, leading to the formation of an exfoliated structure. The trays developed in this study represent an alternative for the packaging of foods with low water contents.