Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility.

Plexins encode receptors for semaphorins, molecular signals guiding cell migration, and axon pathfinding. The mechanisms mediating plexin function are poorly understood. Plexin activation in adhering cells rapidly leads to retraction of cellular processes and cell rounding "cell collapse"). Here we show that, unexpectedly, this response does not require the activity of Rho-dependent kinase (ROCK) nor the contraction of F-actin cables. Interestingly, integrin-based focal adhesive structures are disassembled within minutes upon plexin activation; this is followed by actin depolymerization and, eventually, by cellular collapse. We also show that plexin activation hinders cell attachment to adhesive substrates, blocks the extension of lamellipodia, and thereby inhibits cell migration. We conclude that plexin signaling uncouples cell substrate-adhesion from cytoskeletal dynamics required for cell migration and axon extension.

ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties.

Herpes simplex virus type-1 (HSV1) in which the neurovirulence factor ICP34.5 is inactivated has been shown to direct tumour-specific cell lysis in several tumour models. Such viruses have also been shown to be safe in Phase I clinical trials by intra-tumoral injection in glioma and melanoma patients. Previous work has used serially passaged laboratory isolates of HSV1 which we hypothesized may be attenuated in their lytic capability in human tumour cells as compared to more recent clinical isolates. To produce ICP34.5 deleted HSV with enhanced oncolytic potential, we tested two clinical isolates. Both showed improved cell killing in all human tumour cell lines tested compared to a laboratory strain (strain 17+). ICP34.5 was then deleted from one of the clinical isolate strains (strain JS1). Enhanced tumour cell killing with ICP34.5 deleted HSV has also been reported by the deletion of ICP47 by the up-regulation of US11 which occurs following this mutation. Thus to further improve oncolytic properties, ICP47 was removed from JS1/ICP34.5-. As ICP47 also functions to block antigen processing in HSV infected cells, this mutation was also anticipated to improve the immune stimulating properties of the virus. Finally, to provide viruses with maximum oncolytic and immune stimulating properties, the gene for human or mouse GM-CSF was inserted into the JS1/34.5-/47- vector backbone. GM-CSF is a potent immune stimulator promoting the differentiation of progenitor cells into dendritic cells and has shown promise in clinical trials when delivered by a number of means. Combination of GM-CSF with oncolytic therapy may be particularly effective as the necrotic cell death accompanying virus replication should serve to effectively release tumour antigens to then induce a GM-CSF-enhanced immune response. This would, in effect, provide an in situ, patient-specific, anti-tumour vaccine. The viruses constructed were tested in vitro in human tumour cell lines and in vivo in mice demonstrating significant anti-tumour effects. These were greatly improved compared to viruses not containing each of the modifications described. In vivo, both injected and non-injected tumours showed significant shrinkage or clearance and mice were protected against re-challenge with tumour cells. The data presented indicate that JS1/ICP34.5-/ICP47-/GM-CSF acts as a powerful oncolytic agent which may be appropriate for the treatment of a number of solid tumour types in man.

Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K(D) measured for the binding of wild type and His(6)-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

Clinical characteristics of patients intolerant to VVIR pacing.

The incidence and clinical predictors of the development of intolerance to VVIR pacing have not been extensively studied in prospective long-term randomized trials comparing different pacing modes. The frequency and clinical factors predicting intolerance to ventricular pacing are controversial. The Pacemaker Selection in the Elderly (PASE) Trial enrolled 407 patients aged >/=65 years in a 30-month, single-blind, randomized, controlled comparison of quality of life and clinical outcomes with ventricular pacing and dual-chamber pacing in patients undergoing dual-chamber pacemaker implantation for standard clinically accepted indications. We reviewed the clinical, hemodynamic, and electrophysiologic variables at the time of pacemaker implantation in 204 patients enrolled in the PASE trial and randomized to the VVIR mode, some of whom subsequently required crossover (reprogramming) to DDDR pacing. During a median follow-up of 555 days, 53 patients (26%) crossed over from VVIR to DDDR pacing. A decrease in systolic blood pressure during ventricular pacing at the time of pacemaker implantation (p = 0.001), use of beta blockers at the time of randomization (p = 0.01), and nonischemic cardiomyopathy (p = 0.04) were the only variables that predicted crossover in the Cox multivariate regression model. After reprogramming to the dual-chamber mode, patients showed improvement in all aspects of quality of life, with significant improvements in physical and emotional role. The high incidence of crossover from VVIR to DDDR pacing along with significant improvements in quality of life after crossover to DDDR pacing strongly favors dual-chamber pacing compared with single-chamber ventricular pacing in elderly patients requiring permanent pacing.

Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli.

The construction of new high-copy-number (hcn) lambda-promoter expression vectors is described. All these vectors (1) contain tandem lambda pR and pL promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes, (2) direct expression of the lambda cIts857 gene, enabling their use in any Escherichia coli host strain for thermal induction of gene overexpression, and (3) bear the par locus of plasmid pSC101, ensuring their stable maintenance at hcn in the absence of continuous antibiotic selection. Six of the vectors also contain efficient ribosome-binding sites upstream of unique HpaI or NdeI sites in their MCS regions, and two contain sequences that encode N-terminal poly-His. The performance of these vectors was assessed by using them to overproduce the E. coli HMP flavohaemoprotein and the bacteriophage M13 gene II replicator protein.

An assay for human chorionic gonadotrophin using the capillary fill immunosensor.

Recently there has been much research effort directed towards the development of immunosensors. Optical technologies are currently proving very attractive for the construction of such sensors. The fluorescence capillary fill device (FCFD) has been designed to fulfil these needs. The development of an assay for human chorionic gonadotrophin (hCG) in the FCFD for a variety of body fluids (whole blood, serum, urine and saliva) demonstrates the versatility and assay performance of the device.

Stability of the human nuclear oestrogen receptor: influence of temperature and ionic strength.

The measurement of oestrogen receptors in the nuclei of cells of human breast cancer is becoming increasingly important for patient management. However, the steroid-binding properties of the oestrogen nuclear receptor of human cells under different conditions of temperature and ionic strength have received little attention despite the relevance of the receptor for interpretation of assay data. This paper reports a study on the influence of temperature and ionic strength on the exchange rate of [3H]oestradiol from human breast and endometrial nuclear receptor. When the oestrogen nuclear receptor complex was bound to intact or sheared nuclei, the displacement of bound [3H]oestradiol into buffer containing excess unlabelled oestradiol increased with temperature but was significant over 24 h even at 4 degrees C for nuclei from both breast and endometrium. The use of protease inhibitors combined with relabelling of nuclear receptor after incubation confirmed that the observations at 4 degrees C represented exchange of hormone rather than degradation of the hormone-receptor complex. Degradation was seen at higher temperatures. Measurement of the on-rate of [3H]oestradiol onto nuclear receptor prefilled with unlabelled oestradiol showed that on-rate was also significant over 24 h at 4 degrees C. The displacement of oestradiol from salt-extracted, hydroxylapatite-precipitated receptor also increased with temperature although, in this case, no displacement could be detected until temperatures above 4 degrees C were reached. Only 40% of total oestrogen receptor detectable in intact human nuclei was solubilized by the standard method of salt extraction in 0.6 mol KCl/l. As the salt concentration was raised (0-0.6 mol KCl/l), an increase in the stripping of oestradiol from the hormone-receptor complex was observed. Such stripping of nuclear receptor during salt extraction would lead to a false impression of the proportion of 'empty' nuclear receptors. The results show that filled oestrogen nuclear receptor from human tumour tissue may be assayed by exchange at 4 degrees C over 24 h. This eliminates the protease degradation of receptor which occurs at higher assay temperatures.