MGOUN3: evidence for chromatin-mediated regulation of FLC expression

The MGOUN3(MGO3)/BRUSHY1(BRU1)/TONSOKU(TSK) gene of Arabidopsis thaliana encodes a nuclear leucine-glycine-asparagine (LGN) domain protein that may be implicated in chromatin dynamics and genome maintenance. Mutants with defects in MGO3 display a fasciated stem and disorganized meristem structures. The transition to flowering was examined in mgo3 mutants and it was found that, under short days, the mutants flowered significantly earlier than the wild-type plants. Study of flowering-time associated gene expression showed that the floral transition inhibitor gene FLC was under-expressed in the mutant background. Ectopic expression of the flower-specific genes AGAMOUS (AG), PISTILLATA (PI), and SEPALLATA3 (SEP3) in mgo3 vegetative organs was also detected. Western blot and chromatin immunoprecipitation experiments suggested that histone H3 acetylation may be altered in the mgo3 background. Together, these data suggest that MGO3 is required for the correct transition to flowering and that this may be mediated by histone acetylation and associated changes in FLC expression.     

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.     

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.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.     

Putative involvement of Thioredoxin h in early response to gravitropic stimulation of poplar stems

Gravity perception and gravitropic response are essential for plant development. In herbaceous species, it is widely accepted that one of the primary events in gravity perception involves the displacement of amyloplasts within specialized cells. However, the early signaling events leading to stem reorientation are not fully known, especially in woody species in which primary and secondary growth occur. Thirty-six percent of the identified proteins that were differentially expressed after gravistimulation were established as potential Thioredoxin targets. In addition, Thioredoxin h expression was induced following gravistimulation. In situ immunolocalization indicated that Thioredoxin h protein co-localized with the amyloplasts located in the endodermal cells. These investigations suggest the involvement of Thioredoxin h in the first events of signal transduction in inclined poplar stems, leading to reaction wood formation.     

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly

Background

The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds.

Results

A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed.

Conclusions

By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1196-3) contains supplementary material, which is available to authorized users.     

Ratings of different olfactory judgements in schizophrenia

We assessed the influence of schizophrenia on different olfactory tasks. Forty patients with schizophrenia (20 males and 20 females) and 40 control subjects (20 males and 20 females) were tested. The experiment included two sessions. Initially, 12 odorants were presented at a rate of one per minute. The subjects were asked to rate intensity, pleasantness, familiarity and edibility for each odour using linear rating scales. The odorants were then presented a second time and the subjects were asked to identify them. The results showed that the scores for pleasantness, familiarity, edibility and identification but not intensity were disturbed in patients when compared with control subjects. Furthermore, the familiarity judgement of male patients was more often deficient than that of female patients and they rated odorants as being inedible when the women judged them as neutral. Considered together, these data show that our olfactory test may be used in patients with schizophrenia for evidencing various dysfunctions specific to different types of olfactory processing that represent steps in the odour name identification process.     

Salicylic acid and its location in response to biotic and abiotic stress

Salicylic acid (SA) is an important signal involved in the activation of plant defence responses against abiotic and biotic stress. SA may derive from the phenylpropanoid pathway or via isochorismate synthase as demonstrated in Nicotiana benthamiana, tomato and Arabidopsis thaliana. The phenylpropanoid pathway as well as isochorismate synthase are localized in the chloroplasts but it remains unknown if the end product SA is in the same organelle. We have studied the localization of SA in A. thaliana using the salicylate hydroxylase (NahG) gene expressed with a chloroplast targeting sequence. Plants expressing NahG in the chloroplasts are unable to accumulate SA induced after pathogen or UV exposure. Our data infer that SA is initially located in the chloroplasts.