Retrovirus-polymer complexes: study of the factors affecting the dose response of transduction

We have previously shown that complexes of Polybrene (PB), chondroitin sulfate C (CSC), and retrovirus transduce cells more efficiently than uncomplexed virus because the complexes are large and sediment, reaching the cells more rapidly than by diffusion. Transduction reaches a peak at equal weight concentrations of CSC and PB and declines when the dose of PB is higher or lower than CSC. We hypothesized that the nonlinear dose response of transduction was a complex function of the molecular characteristics of the polymers, cell viability, and the number of viruses incorporated into the complexes. To test this hypothesis, we formed complexes using an amphotropic retrovirus and several pairs of oppositely charged polymers and used them to transduce murine fibroblasts. We examined the effect of the type and concentration of polymers used on cell viability, the size and charge of the complexes, the number of viruses incorporated into the complexes, and virus binding and transduction. Transduction was enhanced (2.5- to 5.5-fold) regardless of which polymers were used and was maximized when the number of positive charge groups was in slight excess (15-28%) of the number of negative charge groups. Higher doses of cationic polymer were cytotoxic, whereas complexes formed with lower doses were smaller, contained fewer viruses, and sedimented more slowly. These results show that the dose response of transduction by virus-polymer complexes is nonlinear because excess cationic polymer is cytotoxic, whereas excess anionic polymer reduces the number of active viruses that are delivered to the cells.     

Specificity and genetic polymorphism in the Vfm quorum sensing system of plant pathogenic bacteria of the genus Dickeya

The Vfm quorum sensing (QS) system is preponderant for the virulence of different species of the bacterial genus Dickeya. The vfm gene cluster encodes 26 genes involved in the production, sensing or transduction of the QS signal. To date, the Vfm QS signal has escaped detection by analytical chemistry methods. However, we report here a strain-specific polymorphism in the biosynthesis genes vfmO and vfmP, which is predicted to be related to the production of different analogues of the QS signal. Consequently, the Vfm communication could be impossible between strains possessing different variants of the genes vfmO/P. We constructed three Vfm QS biosensor strains possessing different vfmO/P variants and compared these biosensors for their responses to samples prepared from 34 Dickeya strains possessing different vfmO/P variants. A pattern of specificity was demonstrated, providing evidence that the polymorphism in the genes vfmO/P determines the biosynthesis of different analogues of the QS signal. Unexpectedly, this vfmO/P-dependent pattern of specificity is linked to a polymorphism in the ABC transporter gene vfmG, suggesting an adaptation of the putative permease VfmG to specifically bind different analogues of the QS signal. Accordingly, we discuss the possible involvement of VfmG as co-sensor of the Vfm two-component regulatory system.     

“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value q_max corresponding to these maxima scaled as C^0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; q_max varied as C^0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 Å were observed with d_f = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.     

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via G_sα were without effect, which was consistent with the localization of G_sα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.     

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantioselectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other. © 1996 Wiley-Liss, Inc.     

Importance of flavonoids in insect--plant interactions: feeding and oviposition

Jeffrey Harborne and colleagues have been responsible for collating the majority of data on the role of flavonoids in insect plant interactions. This article examines some of this information and assesses our knowledge about the role flavonoids play in insect feeding and oviposition behaviour. It is clear that insects can discriminate among flavonoids and that these compounds can modulate the feeding and oviposition behaviour of insects, but further work is required to understand the neural mechanisms associated with these behavioural responses. Despite the wealth of data about the diversity of flavonoids in plants, very few of these compounds have been tested against insects and their role in the evolution of host range in insect--plant interactions has yet to be determined.     

La 12′-hydroxyisostrychnobiline: Nouvel alcaloïde du Strychnos variabilis

The structure of a new bisindole alkaloid, 12′-hydroxyisostrychnobiline, has been proposed from the analysis of its 300 MHz ¹H NMR spectrum and comparison of the spectroscopic data with those of various monomeric and dimeric alkaloids, previously isolated from the same Strychnos species     

A comparative method for finding and folding RNA secondary structures within protein-coding regions

Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins, and evidence is accumulating that this phenomenon may also be found in Eukaryotes. We here present the first comparative method, called RNA-DECODER, which explicitly takes the known protein-coding context of an RNA-sequence alignment into account in order to predict evolutionarily conserved secondary-structure elements, which may span both coding and non-coding regions. RNA-DECODER employs a stochastic context-free grammar together with a set of carefully devised phylogenetic substitution-models, which can disentangle and evaluate the different kinds of overlapping evolutionary constraints which arise. We show that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD.