Design and synthesis of bridged gamma-lactams as analogues of beta-lactam antibiotics

Anti-Bredt bridged bicyclo[3.2.1] gamma-lactams were designed as inhibitors of penicillin binding proteins (PBPs). The compounds were prepared by a carbenoid insertion into a lactam N-H bond. Their weak antibacterial activity could either be explained by a poor chemical stability or by unfavorable steric interactions of the methylene bridge of the gamma-lactam with the targeted enzymes.     

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.     

MHC class I in activity-dependent structural and functional plasticity

Members of the major histocompatibility complex (MHC) class I family of proteins are well known for their central role in the adaptive immune system, where they present self and non-self peptides for immune surveillance. Although the brain has been long considered immune privileged, in part because of an apparent lack of neuronal MHC class I, it has since been shown that MHC class I proteins are expressed by normal, uninfected neurons. Moreover, expression of MHC class I is unusually dynamic in the developing and adult brain, and MHC class I levels in neurons can be regulated by endogenous and exogenous electrical activity. Unexpectedly, several recent studies find that MHC class I is required for distinct activity-dependent events during brain development, adult plasticity, and in response to injury. Together, these studies indicate a novel role for MHC class I proteins in translating electrical activity into changes in synaptic strength and neuronal connectivity in vivo.     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

Transfection with fluorinated lipoplexes based on new fluorinated cationic lipids and in the presence of a bile salt surfactant

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.     

The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain

We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-stranded region F of the knob (residues Leu-485-Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiDeltaHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in vivo.