New solid supports linking nucleoside scaffolds

An easy and efficient strategy to obtain new nucleoside based solid supports in which the nucleoside moieties have been anchored to the solid support through the nucleobase is here proposed. A simple and efficient solid-phase synthesis of 5' and 3'-derivatized uridine analogues has so been developed, following methodologies well established in organic chemistry.     

Vgamma9/Vdelta2 T lymphocytes in Italian patients with Behçet's disease: evidence for expansion,and tumour necrosis factor receptor II and interleukin-12 receptor beta1 expression in active disease

Beh?et's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that gamma/delta T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of gamma/delta T cells with phenotype Vgamma9/Vdelta2, from a group of Italian patients with Beh?et's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vgamma9/Vdelta2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vgamma9/Vdelta2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor beta1 was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor beta1 PE and anti-Vdelta2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vgamma9/Vdelta2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor beta1 expressions were increased in both patients and control individuals. The proportion of Vgamma9/Vdelta2 T cells bearing these receptors was raised in active disease when Vgamma9/Vdelta2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vgamma9/Vdelta2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.     

Use of a novel triple-tracer approach to assess postprandial glucose metabolism

Numerous studies have used the dual-tracer method to assess postprandial glucose metabolism. The present experiments were undertaken to determine whether the marked tracer nonsteady state that occurs with the dual-tracer approach after food ingestion introduces error when it is used to simultaneously measure both meal glucose appearance (R(a meal)) and endogenous glucose production (EGP). To do so, a novel triple-tracer approach was designed: 12 subjects ingested a mixed meal containing [1-(13)C]glucose while [6-(3)H]glucose and [6,6-(2)H(2)]glucose were infused intravenously in patterns that minimized the change in the plasma ratios of [6-(3)H]glucose to [1-(13)C]glucose and of [6,6-(2)H(2)]glucose to endogenous glucose, respectively. R(a meal) and EGP measured with this approach were essentially model independent, since non-steady-state error was minimized by the protocol. Initial splanchnic glucose extraction (ISE) was 12.9% +/- 3.4%, and suppression of EGP (EGPS) was 40.3% +/- 4.1%. In contrast, when calculated with the dual-tracer one-compartment model, ISE was higher (P < 0.05) and EGPS was lower (P < 0.005) than observed with the triple-tracer approach. These errors could only be prevented by using time-varying volumes different for R(a meal) and EGP. Analysis of the dual-tracer data with a two-compartment model reduced but did not totally avoid the problems associated with marked postprandial changes in the tracer-to-tracee ratios. We conclude that results from previous studies that have used the dual-tracer one-compartment model to measure postprandial carbohydrate metabolism need to be reevaluated and that the triple-tracer technique may provide a useful approach for doing so.     

Desialylated N-CAM and chromatin-derived acidic peptide effects in the hypothalamus

Chromatin-derived acidic peptides (ACPs) have been shown to acutely modulate hypothalamic catecholamine release. To investigate whether this effect is mediated through membrane polysialylated neural-cell adhesion molecule (PSA-N-CAM), we pretreated rat hypothalamic synaptosomes with neuraminidase enzyme, which partially cleaves sialic acid residues from N-CAM, and perfused them with ACP-1 (Asp-Asp-Ser-Asp-Glu-Glu-Asn) or a more lipophilic derivative, ACP-2 ([Ala-Ile-Ser-Pro]-Asp-Asp-Ser-Asp-Glu-Glu-Asn). We have found that neuraminidase completely abolish the inhibitory effect of ACP-1 on dopamine release, while the inhibitory activity of ACP-1 on norepinephrine release is partially lost. On the other hand, ACP-2 inhibition of dopamine release is not modified by neuraminidase pretreatment.     

Myogenic potential of mouse primordial germ cells

Primordial germ cells are the only stem cells that retain true developmental totipotency after gastrulation, express markers typical of totipotent/pluripotent status and are able both in vivo and in vitro to give rise to pluripotent stem cells as EC and EG cells. We have therefore explored the possibility of the trans-differentiation of mouse PGCs to a myogenic lineage by transplanting them directly or after in vitro culture into a regenerating muscle and by culturing them on monolayers of differentianting muscle cells. The results obtained suggest that mouse PGCs may trans-differentiate into myogenic cells, provided that their somatic environment is preserved. This occurs at an estimated frequency of 0.01%, which is no higher than that reported for stem cells of adult tissues.     

Functional aspects of protein mono-ADP-ribosylation

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD(+) to acceptor proteins. It is catalysed by cellular ADP-ribosyltransferases and certain bacterial toxins. There are two subclasses of cellular enzymes: the ectoenzymes that modify targets such as integrins, defensin and other cell surface molecules; and the intracellular enzymes that act on proteins involved in cell signalling and metabolism, such as the beta-subunit of heterotrimeric G proteins, GRP78/BiP and elongation factor 2. The genes that encode the ectoenzymes have been cloned and their protein products are well characterized, yet little is known about the intracellular ADP-ribosyltransferases, which may be part of a novel protein family with an important role in regulating cell function. ADP-ribosylation usually leads to protein inactivation, providing a mechanism to inhibit protein functions in both physiological and pathological conditions.     

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27^Kip1 and p16^INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.     

The golden anniversary of cloning: a celebratory essay

May 2002 marked the golden anniversary of the first cloned tadpoles. We celebrate this anniversary, as nuclear transplantation of frog cells into enucleated eggs became the prototype for cloning insects, fish, and mammals. We briefly review the salient results from amphibian cloning. Extension of these studies to mammalian species led to cloning adult cells, important advances in understanding nuclear reprogramming, and the construction of transgenic clones for biomedical applications. In addition, murine cloning clarified two problems unresolved in frog cloning: the unequivocal demonstration that nuclei of fully differentiated cells can direct the formation of fertile adults, and that abnormal expression of genes was responsible for the endoderm and neural syndromes in Rana clones.