Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.     

Simvastatin attenuates expression of cytokine-inducible nitric-oxide synthase in embryonic cardiac myoblasts

Cardiac stem cells or myoblasts are vulnerable to inflammatory stimulation in hearts with infarction or ischemic injury. Widely used for the prevention and treatment of atherosclerotic heart disease, the cholesterol-lowering drugs statins may exert anti-inflammatory effects. In this study, we examined the impact of inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase with simvastatin on the expression of inducible nitric-oxide synthase (iNOS) in embryonic cardiac myoblasts stimulated with the proinflammatory cytokines, interleukin-1 or tumor necrosis factor. Treatment with simvastatin significantly reduced the levels of iNOS mRNA and protein in cytokine-treated rat H9c2 cardiac embryonic myoblasts. Addition of the HMG-CoA reductase product, L-mevalonate, and the by-product of cholesterol synthesis, geranylgeranyl pyrophosphate, could reverse the statin inhibitory effect on iNOS expression. Simvastatin treatment lowered the Rho GTPase activities, whereas the Rho-associated kinase inhibitor Y27632 partially blocked the statin inhibitory effect on nitrite production in the cytokine-treated H9c2 cells. Treatment with simvastatin led to inactivation of NF-kappaB by elevation of the NF-kappaB inhibitor IkappaB and reduction of the NF-kappaB nuclear contents in the cytokine-stimulated H9c2 cells. Hence, treatment with simvastatin can attenuate iNOS expression and NO synthesis in cytokine-stimulated embryonic cardiac myoblasts. The statin inhibitory effect may occur through isoprenoid-mediated intracellular signal transduction, which involves several key signal proteins, such as Rho kinase and IkappaB/NF-kappaB. These data suggest that statin therapy may protect the cardiac myocyte progenitors against the cytotoxicity of cytokine-induced high output of NO production in infarcted or ischemic hearts with inflammation.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.     

Targeting of HLA-DR molecules transduces agonistic functional signals in cutaneous melanoma

The role of human leukocyte antigens (HLA) class II molecules in transducing intracellular signals in immune cells is well established. Solid tumors of different histotype can also express HLA class II antigens; however, their intracellular signaling ability is essentially unknown. Due to the frequent expression of HLA class II molecules in primary and metastatic lesions, cutaneous melanoma was utilized to investigate whether the engagement of HLA-DR molecules transduces functional intracellular signal(s). Triggering of HLA-DR molecules by the anti-HLA-DR monoclonal antibody (mAb) L243 induced a significant (P < 0.05) and dose-dependent growth-inhibition of metastatic melanoma cells Mel 120, as well as their homotypic aggregation. Furthermore, an increase in tyrosine phosphorylation of multiple cellular proteins with a molecular weight ranging from 66 to 130 kD, including p125 focal adhesion kinase, was observed. Lastly, the engagement of HLA-DR molecules by mAb L243 inhibited activator protein-1-DNA binding. Thus, HLA-DR molecules expressed on melanoma cells can transduce functional intracellular signals. This finding is consistent with evidences obtained in hematological malignancies, and suggests the potential usefulness of HLA-DR molecules to set-up new approaches of targeted therapy in metastatic melanoma.     

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.     

Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes

Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.     

Inhibitory effect of ginger (Zingiber officinale) on rat ileal motility in vitro

Ginger (Zingiber officinale rhizome) is a widespread herbal medicine mainly used for the treatment of gastrointestinal diseases, including dyspepsia, nausea and diarrhoea. In the present study we evaluated the effect of this herbal remedy on the contractions induced by electrical stimulation (EFS) or acetylcholine in the isolated rat ileum. Ginger (0.01-1000 microg/ml) inhibited both EFS- and acetylcholine-evoked contractions, being more potent in inhibiting the contractions induced by EFS. The depressant effect of ginger on EFS-induced contractions was reduced by the vanilloid receptor antagonist capsazepine (10(-5) M), but unaffected by the alpha(2)-adrenergic antagonist yohimbine (10(-7) M), the CB(1) receptor antagonist SR141716A (10(-6) M), the opioid antagonist naloxone (10(-6) M) or by the NO synthase inhibitor L-NAME (3 x 10(-4) M). Zingerone (up to 3 x 10(-4) M), one of the active ingredients of ginger, did not possess inhibitory effects. It is concluded that ginger possesses both prejunctional and postjunctional inhibitory effects on ileal contractility; the prejunctional inhibitory effect of ginger on enteric excitatory transmission could involve a capsazepine-sensible site (possibly vanilloid receptors).     

Stochastic optimal therapy for enhanced immune response

Therapeutic enhancement of humoral immune response to microbial attack is addressed as the stochastic optimal control of a dynamic system. Without therapy, the modeled immune response depends upon the initial concentration of pathogens in a simulated attack. Immune response can be augmented by agents that kill the pathogen directly, that stimulate the production of plasma cells or antibodies, or that enhance organ health. Using a generic mathematical model of immune response to the infection (i.e., of the dynamic state of the system), previous papers demonstrated optimal (open-loop) and neighboring-optimal (closed-loop) control solutions that defeat the pathogen and preserve organ health, given initial conditions that otherwise would be lethal [Optimal Contr. Appl. Methods 23 (2002) 91, Bioinformatics 18 (2002) 1227]. Therapies based on separate and combined application of the agents were derived by minimizing a quadratic cost function that weighted both system response and drug usage, providing implicit control over harmful side effects. Here, we focus on the effects that corrupted or incomplete measurements of the dynamic state may have on neighboring-optimal feedback control. Imperfect measurements degrade the precision of feedback adjustments to therapy; however, optimal state estimation allows the feedback strategy to be implemented with incomplete measurements and minimizes the expected effects of measurement error. Complete observability of the perturbed state for this four state example is provided by measurement of four of the six possible pairs of two variables, either set of three variables, or all four variables. The inclusion of state estimation extends the applicability of optimal control theory for developing new therapeutic protocols to enhance immune response.