A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Nitric oxide plays a central role in determining lateral root development in tomato

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological processes in plants, most of them involving cross-talk with traditional phytohormones. Auxin is the main hormone that regulates root system architecture. In this communication we report that NO promotes lateral root (LR) development, an auxin-dependent process. Application of the NO donor sodium nitroprusside (SNP) to tomato (Lycopersicon esculentum Mill.) seedlings induced LR emergence and elongation in a dose-dependent manner, while primary root (PR) growth was diminished. The effect is specific for NO since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) blocked the action of SNP. Depletion of endogenous NO with CPTIO resulted in the complete abolition of LR emergence and a 40% increase in PR length, confirming a physiological role for NO in the regulation of root system growth and development. Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed that the NO signal was specifically located in LR primordia during all stages of their development. In another set of experiments, SNP was able to promote LR development in auxin-depleted seedlings treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Moreover, it was found that LR formation induced by the synthetic auxin 1-naphthylacetic acid (NAA) was prevented by CPTIO in a dose-dependent manner. All together, these results suggest a novel role for NO in the regulation of LR development, probably operating in the auxin signaling transduction pathway.Abbreviations CPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - DAF-2 DA 4,5-Diaminofluorescein diacetate - LR Lateral root - NAA 1-Naphthylacetic acid - NO Nitric oxide - NPA N-1-Naphthylphthalamic acid - PR Primary root - SNP Sodium nitroprusside     

A fragment of chloroplast DNA was transferred horizontally,probably from non-eudicots,to mitochondrial genome of <Emphasis Type="Italic">Phaseolus</Emphasis>

The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnAfragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabalessequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants.     

Diurnal rhythms of pinealocyte ultrastructure, pineal serotonin content and plasma melatonin level in the domestic pig

The study was conducted to investigate diurnal changes in pinealocyte ultrastructure, pineal serotonin content and plasma melatonin concentration in the domestic pig. The immature pigs (n=24) were kept under a cycle of 12 h light : 12 h dark, with a photophase between 0800 and 2000. During the photophase the animals were exposed to direct sunlight. After four weeks the gilts were slaughtered at 0900, 1400, 2100 and 0200. The pineals were removed and divided into two parts - one for quantitative ultrastructural study (by a point count method) and one for serotonin assay. Simultaneously, blood samples were taken for melatonin assay. The relative volume of mitochondria in pinealocyte perikarya was significantly higher at 1400 than at 0200 and 0900 as well as at 2100 than at 0200. The relative volume of Golgi apparatus was higher at 0900 and 1400 than at 0200. The relative volume of dense bodies of the MBB-1 type in pinealocyte perikarya was significantly lower at 1400 and 2100 than at 0900. In contrast, the relative volume of MBB-2 was higher at 1400 than at 0900 and 0200. The numerical density of DCV in perikarya was significantly higher at 0200 than at 1400. No significant differences were found in rough endoplasmic reticulum, lysosomes and multivesicular bodies. The pineal serotonin content showed a prominent rhythm with the maximum at 1400. The plasma melatonin concentration was significantly higher at 0200 than at 0900, 1400 and 2100. The obtained results demonstrate that both pinealocyte ultrastructure and pineal biochemistry in the pig undergo significant changes in the course of the diurnal rhythm.     

First cleavage of the mouse embryo responds to change in egg shape at fertilization

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.     

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with -glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.     

Sera of lung cancer patients affect the release of Th1, Th2 and monocyte-derived cytokines, and the expression of IL-2Ralpha by normal, stimulated mononuclear cells

We have shown that the sera of lung cancer patients affect the response of ConA-stimulated normal peripheral blood mononuclear cells by decreasing the expression of IL-2Ralpha and inhibiting the release of IL-1beta and IL-2. A tendency to enhance the release of IL-6 was also observed. We conclude that an imbalance in the Th1/Th2 cytokine response, typical for cancer patients, may at least partly be related to soluble factors circulating in the patients' blood. We discuss a putative role of serum IL-10, IL-1ra, and soluble IL-2Ralpha in the effects observed.     

Directing pluripotent cell differentiation using "diced RNA" in transient transfection

Embryonic stem (ES) and embryonic carcinoma (EC) cells are pluripotent and have the capacity to differentiate into many cell types. The ability to direct their differentiation should have considerable practical applications. Here, we first report the use of diced short interfering RNAi against Oct4 in a transient approach, to direct differentiation of ES towards the trophectoderm lineage. We then apply this approach to downregulate Smad4 in mouse P19 EC cells. We have found that this leads to an increase in the levels of Pax6 (a neuroectoderm marker), reduction in the levels of Brachyury (a mesoderm marker), and a 3-fold increase in the number of betaIII tubulin-positive colonies when these cells were allowed to differentiate. This indicates a redirection of cell fate towards the neuroectoderm lineage. Thus, transient RNAi could provide a valuable tool to direct pluripotent cells along specific pathways of differentiation while circumventing permanent genetic changes.