Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.     

Non-thiol farnesyltransferase inhibitors: N-(4-Acylamino-3-benzoylphenyl)-4-nitrocinnamic acid amides.

We have developed the 4-nitrocinnamoyl substituted benzophenone 4a as a novel non-thiol farnesyltransferase inhibitor. Replacement of the p-tolyl moiety of our initial lead structure 4a by different para and ortho substituted phenyl residues as well as by 1-naphthyl resulted in derivatives with considerably enhanced activity displaying IC(50) values between 42 and 52 nM. These compounds represent novel, readily accessible non-thiol farnesyltransferase inhibitors being more active than the corresponding thiol-containing analogues.     

Long-term effects of Ca2+ on structure and contractility of vascular smooth muscle

Culture of dispersedsmooth muscle cells is known to cause rapid modulation from thecontractile to the synthetic cellular phenotype. However, organ cultureof smooth muscle tissue, with maintained extracellular matrix andcell-cell contacts, may facilitate maintenance of the contractilephenotype. To test the influence of culture conditions, structural,functional, and biochemical properties of rat tail arterial rings wereinvestigated after culture. Rings were cultured for 4 days in theabsence and presence of 10% FCS and then mounted for physiologicalexperiments. Intracellular Ca²⁺concentration([Ca²⁺]_i)after stimulation with norepinephrine was similar in rings culturedwith and without FCS, whereas force development after FCS was decreasedby >50%. The difference persisted after permeabilization with-escin. These effects were associated with the presence ofvasoconstrictors in FCS and were dissociated from itsgrowth-stimulatory action. FCS treatment increased lactate productionbut did not affect ATP, ADP, or AMP contents. The contents of actin andmyosin were decreased by culture but similar for all cultureconditions. There was no effect of FCS on calponin contents or myosinSM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration notfound after culture without FCS or with FCS + verapamil (1 µM) tolower[Ca²⁺]_i.The decreased force-generating ability after culture with FCS is thusassociated with increased[Ca²⁺]_iduring culture and not primarily caused by growth-associated modulationof cells from the contractile to the synthetic phenotype.

     

Cloning,expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a relative molecular mass of 25 000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein. Correspodence to: A. J. Salerno     

Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis

Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF₅₀₈ chromosomes. Three novel mutations,-471del3 (5 flanking region), 3171insC (exon 17a) and 4700(T)_8/9 (3 non-translated region) of the CFTR gene were found. Mutation 3171insC occured in conjunction with the delta F₅₀₈ mutation on the other allele of a child presenting with severe pathology. Mutation -471 del3 has so far only been found in one healthy individual and her father, and 4700(T)_8/9 is a DNA sequence polymorphism.     

Spatio‐temporal analysis of Nova virus,a divergent hantavirus circulating in the European mole in Belgium

Over the last decade, the recognized host range of hantaviruses has expanded considerably with the discovery of distinct hantaviruses in shrews, moles and bats. Unfortunately, in‐depth studies of these viruses have been limited. Here we describe a comprehensive analysis of the spatial distribution, genetic diversity and evolution of Nova virus, a hantavirus that has the European mole as its natural host. Our analysis demonstrated that Nova virus has a high prevalence and widespread distribution in Belgium. While Nova virus displayed relatively high nucleotide diversity in Belgium, amino acid changes were limited. The nucleocapsid protein was subjected to strong purifying selection, reflecting the strict evolutionary constraints placed upon Nova virus by its host. Spatio‐temporal analysis using Bayesian evolutionary inference techniques demonstrated that Nova virus had efficiently spread in the European mole population in Belgium, forming two distinct clades, representing east and west of Belgium. The influence of landscape barriers, in the form of the main waterways, on the dispersal velocity of Nova virus was assessed using an analytical framework for comparing Bayesian viral phylogenies with environmental landscape data. We demonstrated that waterways did not act as an environmental resistance factor slowing down Nova virus diffusion in the mole population. With this study, we provide information about the spatial diffusion of Nova virus and contribute sequence information that can be applied in further functional studies.     

Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.