Gene transfer into skeletal muscle using novel AAV serotypes

BACKGROUND: Skeletal muscle is an interesting target for gene delivery because of its mass and because the vectors can be delivered in a noninvasive way. Adeno-associated virus (AAV) vectors are capable of transducing skeletal muscle fibers and achieving stable and safe transgene expression. To date, most animal experiments using AAV have been based on AAV serotype 2, but some recent studies have demonstrated that AAV1 is more efficient than AAV2/2 in transducing muscle fibers. Recently, novel AAVs (AAV7 and AAV8) were isolated from rhesus macaques. METHODS: We injected three different muscles (gastrocnemius, soleus, biceps femoris) of immunocompetent C57BL/6 mice with different pseudotyped AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8) and quantitatively compared the different gene transfer efficiencies. RESULTS: The efficiencies of transduction in skeletal muscle with AAV2/7 and AAV2/8 were similar to AAV2/1, and higher than that seen with AAV2/2 and AAV2/5. All serotypes were able to transduce both slow and fast muscle fibers similarly at the vector titer used (1x10(11) genome copies per mouse). Despite a limited inflammatory response (slightly higher when using AAV2/2, AAV2/7 and AAV2/8 vectors than AAV2/1 and AAV2/5), transgene expression was observed throughout the length of the experiment. DISCUSSION: These results show that AAV2/7 and AAV2/8 are able to transduce muscle fibers of immunocompetent mice very efficiently, offering new perspectives in gene transfer of skeletal muscle.     

Safety of TNF-blocking agents in rheumatic patients with serology suggesting past hepatitis B state: results from a cohort of 21 patients

Introduction  

Reactivation of hepatitis B virus (HBV) infection in patients with past infection has been described in 5% to 10% of individuals undergoing immunosuppressive therapies. No data are available to date on the outcome of patients treated by tumour necrosis factor-alpha (TNFα) inhibitors for chronic arthritis with a serological pattern of past HBV infection. The aim of our study was to monitor HBV markers in HBV surface antigen (HBsAg)-negative/anti-HBcAb-positive patients treated with a TNFα inhibitor for inflammatory arthritides.     

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features

The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C(HCV)169) and its N-terminal region from positions 1 to 117 (C(HCV)117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C(HCV)169 and C(HCV)117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C(HCV)169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of alpha-helices (50%). In contrast, C(HCV)117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.     

Heavy metals in sewage sludges contribute to their adverse effects on the arbuscular mycorrhizal fungus<Emphasis Type="Italic">Glomus mosseae</Emphasis>

Applying sewage sludges to agricultural land is a widespread practice because of the sludges’ agronomic value as a source of plant nutrients and organic matter. Nevertheless, sludges often contain micropollutants that can constitute a menace for health and the environment. Arbuscular mycorrhizal fungi are sensitive to sewage sludges that have been spiked, or not, with metallic trace elements (MTE). Here we have investigated if MTE in sewage sludges could be responsible for effects on mycorrhizal development betweenGlomus mosseae andMedicago truncatula. The impact of a dehydrated or composted urban sewage sludge spiked or not with MTE, was tested on spore germination and root colonization byG. mosseae. The sewage sludges depressed both the presymbiotic andin planta stages of development of the mycorrhizal fungus. This negative effect was more related to the metallic pollutant contents of the sludges than to the presence of antagonistic microorganisms or phosphorus.     

Amphiphilic and Nonamphiphilic Forms of Torpedo Cholinesterases: I. Solubility and Aggregation Properties

We report an analysis of the solubility and hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X-100 is approximately 2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE-depleted Triton X-100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which is also detectable in detergent-soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low-salt-soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).     

Changes in the initial phase of lipid peroxidation induced by elicitor from Phytophthora infestans in Solanum species

The initial phase of the lipid peroxidation process in leaves of Solanum nigrum var. gigantea, Solanum tuberosum cv Bzura and clone H-8105, which represent non-host resistance, field resistance and susceptibility, respectively, against Phytophthora infestans, was investigated. Based on quantitative and qualitative high-performance liquid chromatography (HPLC) analyses of free and esterified fatty acid hydroperoxides (FAHs), we characterized the lipid peroxidation process induced by the pathogen-derived elicitor, culture filtrate (CF), in leaves of the studied genotypes. In all plants, FAHs generated due to 13-lipoxygenase (LOX) action dominated over those from the non-enzymatic pathway. The FAHs derived from 9-LOX activity were found only in CF-treated leaves of the non-host resistant S. nigrum. However, experiments in vitro and in planta with exogenous linoleic acid (LA) as a substrate for LOX revealed high constitutive activity of 9-LOX in all genotypes, which increased in response to CF treatment. The time course changes in polyunsaturated fatty acid (PUFA) pools in the total lipid fractions as well as the degree of their oxidation suggested that CF-induced PUFA peroxidation was enhanced mostly in S. nigrum, less so in Bzura and least in the susceptible clone H-8105. The obtained results are discussed in light of the overall biochemical cell status of plants in the studied interactions.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.