Mutation of SOCS2 induces structural and functional changes in mammary development.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.

Electrokinetic properties of healthy and ß-thalassemia erythrocyte membranes under in vitro exposure to static magnetic field.

Introduction: The current understanding of the biological impacts of a static magnetic field (SMF) is restricted to the direct interactions of the magnetic field with biological membranes. The electrokinetic (zeta) potential is an electrochemical property of erythrocyte surfaces which was negatively charged in physiological media after SMF exposure (0.1‒2.0 T). Methods: The novel data about electrokinetic parameters of the erythrocytes is determined by microelectrophoresis after SMF-exposure in norm and heterozygous ß-thalassemia. The methods of light scattering, lipid peroxidation, fluorescence microscopy are used. Results: The electrokinetic potential of erythrocytes in norm is increased after SMF intensities due to enhanced negatively exposed charges on the outer surface of the membrane accompanied by an increase in light scattering where changes in cell morphology are observed. Conversely, a decrease in the zeta potential of ß-thalassemia erythrocytes upon SMF-treatment was determined because of the reduction in the surface electrical charge of the membranes, where a significant decrease in light scattering at 1.5 T and 2.0 T was recorded. Exposure to SMF (0.5-2.0 T) was associated with an increase in the malondialdehyde content in erythrocytes. Biophysical studies regarding the influence of SMF on the electrostatic free energy of cells shows an increase in negative values in healthy erythrocytes, which corresponds to the implementation of a spontaneous process. This is also the process in ß-thalassemia cells after SMF exposure with lower negative values of free electrostatic energy than erythrocytes in norm. Discussion: The effect of static magnetic field (SMF 0.1-2.0 T) on the electrokinetic and morphological characteristics of erythrocytes in norm and ß-thalassemia is determined and correlated with the increase/reduction in surface charge and shrinkage/swelling of the cells, respectively. Lipid peroxidation of healthy and ß-thalassemia erythrocytes caused an enhancement of lipid peroxidation because of the higher concentrations of TBARS products in cellular suspension. SMF (0.1‒2.0 T) altered the spontaneous chemical processes with negative values of electrostatic free energy of erythrocytes in norm and ß-thalassemia accompanied by a lower FITC-Concanavalin A binding affinity to membrane receptors (SMF 2.0 T). The electrokinetic properties of human erythrocytes in norm and ß-thalassemia upon SMF treatment and their interrelationship with the structural-functional state of the membrane were reported. The presented work would have future fundamental applications in biomedicine.

Epigenetics: New Insights into Mammary Gland Biology.

The mammary gland undergoes important anatomical and physiological changes from embryogenesis through puberty, pregnancy, lactation and involution. These steps are under the control of a complex network of molecular factors, in which epigenetic mechanisms play a role that is increasingly well described. Recently, studies investigating epigenetic modifications and their impacts on gene expression in the mammary gland have been performed at different physiological stages and in different mammary cell types. This has led to the establishment of a role for epigenetic marks in milk component biosynthesis. This review aims to summarize the available knowledge regarding the involvement of the four main molecular mechanisms in epigenetics: DNA methylation, histone modifications, polycomb protein activity and non-coding RNA functions.

Structure, regulation, and pharmacological modulation of PP2A phosphatases.

Protein phosphatases of the type 2A family (PP2A) represent a major fraction of cellular Ser/Thr phosphatase activity in any given human tissue. In this review, we describe how the holoenzymic nature of PP2A and the existence of several distinct PP2A composing subunits allow for the generation of multiple structurally and functionally different PP2A complexes, explaining why PP2A is involved in the regulation of so many diverse cell biological and physiological processes. Moreover, in human disease, most notably in several cancers and Alzheimer's Disease, PP2A expression and/or activity have been found significantly decreased, underscoring its important functions as a major tumor suppressor and tau phosphatase. Hence, several recent preclinical studies have demonstrated that pharmacological restoration of PP2A activity, as well as pharmacological PP2A inhibition, under certain conditions, may be of significant future therapeutic value.

Surface electric charge of thylakoid membranes from genetically modified tobacco plants under freezing stress.

In the present study we characterize for the first time electrokinetic and light scattering properties of thylakoids from freezing-tolerant tobacco plants, transformed to accumulate osmoprotectants (proline: AtP5Cs, VacP5Cs; fructan: SacB; glycine betaine: codA). Tobacco plants of wild type (WT) and transformed variants were cultivated at 2°C (cold acclimated) and -2°C (freezing stressed). "Lower salt" thylakoids (I=0.0006) of WT and SacB plants exhibited a decrease in electrophoretic mobility (EPM) after (2°C) treatment. AtP5Cs thylakoids (22°C) show a substantial increase in negative electrical charge (σ) upon illumination. We observed that "low salt"SacB thylakoids at 22°C and 2°C increased the σ on their membrane surfaces during the process of acclimation. WT (22°C) and AtP5Cs thylakoids (2°C) in "low salt" media (I=0.0156) showed a substantial increase in surface electrical charge upon illumination. Cold acclimation on WT and freezing stress on transformed plants resulted in a decrease in aggregation of thylakoids at both ionic strengths. There was a large enhancement in the relaxation capacity of reverse photosynthetic reactions in codA and SacB tobacco after freezing stress. Maximal intensity of the delayed light emission following low temperature stimuli was decreased, revealing a path for tobacco transformants to improve their cold stress tolerance. Here, we suggest the EPM value as an indicator for stability of thylakoids undergone genetic transformation.

The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity.

Protein phosphatase type 2A (PP2A) enzymes constitute a large family of Ser/Thr phosphatases with multiple functions in cellular signaling and physiology. The composition of heterotrimeric PP2A holoenzymes, resulting from the combinatorial assembly of a catalytic C subunit, a structural A subunit, and regulatory B-type subunit, provides the essential determinants for substrate specificity, subcellular targeting, and fine-tuning of phosphatase activity, largely explaining why PP2A is functionally involved in so many diverse physiological processes, sometimes in seemingly opposing ways. In this review, we highlight how PP2A holoenzyme biogenesis and enzymatic activity are controlled by a sophisticatedly coordinated network of five PP2A modulators, consisting of α4, phosphatase 2A phosphatase activator (PTPA), leucine carboxyl methyl transferase 1 (LCMT1), PP2A methyl esterase 1 (PME-1) and, potentially, target of rapamycin signaling pathway regulator-like 1 (TIPRL1), which serve to prevent promiscuous phosphatase activity until the holoenzyme is completely assembled. Likewise, these modulators may come into play when PP2A holoenzymes are disassembled following particular cellular stresses. Malfunctioning of these cellular control mechanisms contributes to human disease. The potential therapeutic benefits or pitfalls of interfering with these regulatory mechanisms will be briefly discussed.

Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.