MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.     

Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.     

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK)

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.     

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.     

Structural investigations on the Nodal‐Cripto binding: A theoretical and experimental approach

Nodal, a member of the transforming growth factor‐β superfamily, is a potent embryonic morphogen also implicated in tumor progression. Up to date structural information on the interaction of Nodal with its molecular partners are unknown. To deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto‐dependent tumor progression, we present here a molecular model of activin receptor‐like kinase 4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal, which encompasses residues 43–69 and includes the prehelix loop and the H3 helix. This hypothesis has been subsequently assessed by surface plasmon resonance binding assays between the full‐length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition, the binding affinity between the full‐length Nodal and Cripto proteins has been evaluated for the first time. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1011–1021, 2010.     

Seed morphology in some European aconites (Aconitum,Ranunculaceae)

The seed coat morphology, investigated in taxa representative of the main European groups ofAconitum, are in good agreement with the current taxonomy of the genus. The seed coat microcharacteristics (warty epidermal cells) are very constant. There is a trend for the reduction of longitudinal wings on the edges concomitant with the development of ridges and transverse wings on the faces. Another morphological progression leads from smooth to rugulose and eventually to transverse wing-bearing seed faces. A working hypothesis suggests an ecological adaptative significance to these changes.     

The Cannabinoid Receptor Type 2 as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.     

Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.