Renata Battini

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.

TGFbeta/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Wnt or TGFβ/BMP. Interestingly, mesoangioblasts express receptors of the TGFβ/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFβ and into osteoblasts in response to BMP. In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Wnts and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, even after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

Glutamatergic synaptic responses and long‐term potentiation are impaired in the CA1 hippocampal area of calbindin D28k‐deficient mice

The contribution of the cytosolic calcium binding protein calbindin D28K (CaBP) to glutamatergic neurotransmission and synaptic plasticity was investigated in hippocampal CA1 area of wild‐type and antisense transgenic CaBP‐deficient mice, with the use of extracellular recordings in the ex vivo slice preparation. The amplitude of non‐N‐methyl‐D‐aspartate receptor (non‐NMDAr)‐mediated extracellular field excitatory postsynaptic potentials (fEPSPs) recorded in control medium was significantly greater in CaBP‐deficient mice, whereas the afferent fiber volley was not affected. In contrast, the amplitude of NMDAr‐mediated fEPSPs isolated in a magnesium‐free medium after blockade of non‐NMDAr and GABAergic receptors was significantly depressed in these animals. No alteration in the magnitude of paired‐pulse facilitation was found, indicating that the presynaptic calcium mechanisms controlling glutamate release were not altered in CaBP‐deficient mice. The magnitude and time course of the short‐term potentiation (STP) of fEPSPs induced by a 30 Hz conditioning stimulation, which was blocked by the NMDAr antagonist 2‐amino‐5‐phosphonovalerate acid (2‐APV), was not impaired in the transgenic mice, whereas long‐term potentiation (LTP) induced by a 100 Hz tetanus was not maintained. The long‐term depression (LTD) induced by low‐frequency stimulation (1 Hz, 15 min) in the presence of the GABA antagonist bicuculline was not altered. These results argue for a contribution of CaBP to the mechanisms responsible for the maintenance of long‐term synaptic potentiation, at least in part by modulating the activation of NMDA receptors. Synapse 33:172–180, 1999.

Cationic Liposomes for Gene Transfection

Cationic liposomes spontaneously interact with the negatively charged DNA to form a stable complex that promotes the gene transfer to cells. The mode of formation and the size of cationic liposomes/DNA complexes were investigated using the atomic force microscopy (AFM). Also the most important physical–chemical factors involved in cationic liposome-mediated gene transfection, e.g. size and lipidic composition, were evaluated through the transfection of complexes with different liposomes/DNA molar ratio into three types of cultured cells. Cationic liposomes, composed of a neutral lipid (phosphatidilcoline), a cationic lipid dimethyldioctadecylammonium bromide (DDAB), a co-lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and a phospholipid derivative of polyethylene glycol (DSPE-mPEG) at different molar ratio, were mixed with a plasmid pCMVβ to form liposomes/DNA complexes. We have demonstrated that the complexes were made by complicated structures in which the liposomes tend to aggregate and the DNA is surrounded by lipidic material. In vitro transfection efficiency by liposomes/plasmid pCMVβ complexes was found to depend on the kind of lipid associated in the liposomes and the liposomes/DNA mixing ratio. The importance of associating DOPE in cationic liposomes was confirmed; this co-lipid is able to improve the ability of cationic liposomes to transfect cells but in addition, the AFM images and the EtBr fluorescence experiments have suggested that this lipid can also play an important role to facilitate the formation of stable liposomes, which efficaciously protect the DNA by nuclease digestion.

Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition

The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14–3–3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.

Differentiation-dependent expression of apolipoprotein A-I in chicken myogenic cells in culture☆

Northern blot hybridization experiments showed that Apolipoprotein A-I (Apo A-I) mRNA is present at high concentration in chicken myotubes cultured in vitro, while it is virtually absent in fibroblasts and myoblasts. Myotubes are also capable of translating and secreting in the culture medium a protein which is specifically immunoprecipitated by anti-Apo A-I antibodies and has the same electrophoretic mobility as Apo A-I purified from circulating high-density lipoproteins. The appearance of Apo A-I mRNA in myotubes depends on the transcriptional activation of the corresponding gene, as it was shown by hybridizing 32P-labeled RNA synthesized in isolated nuclei to Apo A-I cDNA. The activation of the Apo A-I gene is regulated by the muscle cell coordinately with muscle-specific genes. In fact, treatment with TPA, a powerful inhibitor of differentiation, efficiently prevents myoblasts from producing Apo A-I mRNA, as well as muscle actin mRNA, and causes myotubes to quickly cease Apo A-I mRNA synthesis. The existence of a strict relationship between Apo A-I mRNA concentration and myogenic cell differentiation was also confirmed by experiments with quail myoblasts transformed with a temperature-sensitive mutant of the Rous Sarcoma Virus. Cells raised at the permissive temperature (undifferentiated phenotype) do not contain Apo A-I as well as alpha-actin mRNAs, while shifting to the nonpermissive temperature (differentiated phenotype) causes a rapid increase in Apo A-I and alpha-actin mRNA concentration.

Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts.

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function, we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.