Vav1 modulates protein expression during ATRA-induced maturation of APL-derived promyelocytes: a proteomic-based analysis

Overexpression of Vav1 promotes the overcoming of the differentiation blockade that characterizes acute promyelocytic leukemia cells. At variance, down-modulation of Vav1 prevents ATRA-induced maturation, and in particular, the inhibition of its tyrosine phosphorylation prevents the neutrophil differentiation-related changes of cell morphology. These findings allowed to identify Vav1 as a crucial protein in the ATRA-dependent differentiation of tumoral promyelocytes. By means of a proteomic approach, here we have investigated a possible role for Vav1 in modulating protein expression during ATRA treatment of tumoral promyelocytes. We have performed high-resolution 2-DE coupled with mass spectra analysis of HL-60 and NB4 promyelocytic cell lines induced to differentiate with ATRA when the amounts or the tyrosine phosphorylation of Vav1 were forcedly reduced. We have found that the down-regulation of Vav1 affects the expression level of a number of proteins, including cell cycle/apoptosis- and cytoskeleton-related proteins. In particular, the expression of 14-3-3epsilon, alpha-enolase, alpha-tubulin and splice isoform 2 of alpha3 proteasome subunit changed as a consequence of the down-modulation of Vav1 during the differentiation of both HL-60 and NB4 cell lines, suggesting that these proteins may constitute a common part of the ATRA-induced pathway during maturation of APL-derived promyelocytes. These results indicate an unprecedented role for Vav1 in the maturation of myeloid cells as a regulator of protein expression.     

An automatic translation of SBML into Beta-binders

A translation of Systems Biology Markup Language (SBML) into a process algebra is proposed in order to allow the formal specification, the simulation and the formal analysis of biological models. Beta-binders, a language with a quantitative stochastic extension, is chosen for the translation. The proposed translation focuses on the main components of SBML models, as species and reactions. Furthermore, it satisfies the compositional property, i.e. the translation of the whole model is obtained by composing the translation of the subcomponents. An automatic translator tool of SBML models into Beta-binders has been implemented as well. Finally, the translation of a simple model is reported.     

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

Background

The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.

Methods

In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.

Results

Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).

Conclusion

These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.     

Poly(amidoamine) conjugates with disulfide-linked cholesterol pendants self-assembling into redox-sensitive nanoparticles

Poly(amidoamine) (PAA) networks that are obtained by the use of cystamine as a cross-linking agent in the reaction with 2,2'-dithiodipyridine turn into linear PAAs with dithiopyridyl side groups that easily undergo an exchange reaction with thiocholesterol. The resultant products represent the first examples of amphiphilic PAA-cholesterol conjugates in which lipophilic cholesterol moieties are linked to the hydrophilic PAA chain by S-S bonds that are stable in blood but cleavable inside cells. In aqueous media, these conjugates self-assemble into nanoaggregates whose inner cores consist of lipophilic cholesterol domains. A series of PAA-cholesterol conjugates that are derived from two different bis-acrylamides, namely 2,2-bis(acrylamido)acetic acid and 1,4-bis(acryloyl)piperazine, and that have different cholesterol contents were obtained. All products were characterized by (1)H and (13)C NMR spectroscopy, and the average molecular weights of the soluble polymers were determined by size exclusion chromatography. In all instances, the segregation of cholesterol residues from the aqueous medium was revealed by the comparison of their NMR spectra in CDCl3 and D2O, respectively. The TEM analysis of the PAA-cholesterol aggregates in aqueous buffers revealed homogeneous round nanospheres whose dimensions and dimension distributions were determined by DLS. Preliminary cytocompatibility tests demonstrated that all prepared PAA-cholesterol samples are cytocompatible and thus show potential for biotechnological applications.     

Clines in clock genes: fine-tuning circadian rhythms to the environment

The dissection of the circadian clock into its molecular components represents the most striking and well-studied example of a gene regulatory network underlying a complex behavioural trait. By contrast, the evolutionary analysis of the clock has developed more slowly. Here we review studies that have surveyed intraspecific clock gene variation over large geographical areas and have discovered latitudinal clines in gene frequencies. Such spatial patterns traditionally suggest that natural selection shapes genetic variation, but it is equally possible that population history, or a mixture of demography and selection, could contribute to the clines. We discuss how population genetics, together with functional assays, can illuminate these possible cases of natural selection in Drosophila clock genes.     

In vitro cytotoxic activity of Salsola oppositifolia Desf. (Amaranthaceae) in a panel of tumour cell lines

The aim of the present study was to evaluate for the first time the in vitro cytotoxic activity of fractions and isolated flavonols from Salsola oppositifolia Desf. (Amaranthaceae). The n-hexane fraction demonstrated an effective cytotoxic activity on the large lung carcinoma and amelanotic melanoma cell lines with IC50 values of 19.1 microg/ml and 24.4 microg/ml, respectively. Also the dichloromethane fraction exhibited cytotoxic activity against COR-L23 (IC50 30.4 microg/ml) and C32 (IC50 33.2 microg/ml) cells, while the EtOAc fraction demonstrated a selective cytotoxic activity against MCF-7 cells (IC50 67.9 microg/ml). The major active constituents of this fraction were isorhamnetin-3-O-glucoside (1) and isorhamnetin-3-O-rutinoside (2), which showed an interesting activity against the cell line MCF-7 with IC50 values of 18.2 and 25.2 microg/ml, respectively. Compound 2 exhibited a strong activity against the hormone-dependent prostate carcinoma LNCaP cell line with an IC50 of 20.5 microg/ml. Constituents of S. oppositifolia were identified by GC-MS and NMR analyses.     

2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors

The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoroacetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroacetyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form.     

Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog

The post-translational modification of proteins by the covalent attachment of carbohydrates to specific side chains, or glycosylation, is emerging as a crucial process in modulating the function of proteins. In particular, the dynamic processing of the oligosaccharide can correlate with a change in function. For example, a potent macrophage-activating factor, Gc-MAF, is obtained from serum vitamin D binding protein (VDBP) by stepwise processing of the oligosaccharide attached to Thr 420 to the core alpha-GalNAc moiety. In previous work we designed a miniprotein analog of Gc-MAF, MM1, by grafting the glycosylated loop of Gc-MAF on a stable scaffold. GalNAc-MM1 showed native-like activity on macrophages (Bogani 2006, J. Am. Chem. Soc. 128 7142-43). Here, we present data on the thermodynamic stability and conformational dynamics of the mono- and diglycosylated forms. We observed an unusual trend: each glycosylation event destabilized the protein by about 1 kcal/mol. This effect is matched by an increase in the mobility of the glycosylated forms, as evaluated by molecular dynamics simulations. An analysis of the solvent-accessible surface area shows that glycosylation causes the three-helix bundle to adopt conformations in which the hydrophobic residues are more solvent exposed. The number of hydrophobic contacts is also affected. These two factors, which are ultimately explained with a change in occupancy for conformers of specific side chains, may contribute to the observed destabilization.