Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E(2) synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE(2) synthase-1 (mPGES-1) isomerizes COX-2-derived PGH(2) to PGE(2). Here, we evaluated the effect of mPGES-1-deficiency on the nociceptive behavior in various models of nociception that depend on PGE(2) synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE(2) synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE(2) synthesis to PGD(2), PGF(2alpha) and 6-keto-PGF(1alpha) (stable metabolite of PGI(2)). Since the latter prostaglandins serve also as mediators of nociception they may compensate the loss of PGE(2) synthesis in mPGES-1-deficient mice.     

Growth hormone releasing effect of hpGRF-40 in rats at different time intervals following ablation of the mediobasal hypothalamus

We have investigated the effect of hypothalamo-pituitary disconnection in the rat on the growth hormone (GH) responsiveness to human pancreatic GH-releasing factor (hpGRF). Adult female rats, sham-operated (sham-op) or bearing a complete mechanical ablation of the mediobasal hypothalamus (MBH-A) were challenged, while under urethane anesthesia, with hpGRF-40 (20,100,500 ng/rat i.v.) at different time intervals after surgery. In sham-op rats only 500 ng/rat of hpGRF-40 stimulated GH release, while in 1-and 7-day MBH-A rats the stimulation also occurred with the lower hpGRF doses and the rise in plasma GH was greater than in sham-op controls. Twenty-one and 42 days after the placing of the lesions the GH response to hpGRF-40 was still present at the 500 ng/rat dose, though it was smaller than in sham-op controls. Evaluation of pituitary GH content demonstrated a progressive and rapid decline starting the first day after the placing of the lesions. These data indicate that GH responsiveness to hpGRF is: 1) enhanced in the anterior pituitary shortly after hypothalamo-pituitary disconnection and, 2) despite a striking reduction of the pituitary GH stores, it is maintained after these lesions.The physiologic growth hormone (GH) releaser in the rat is GH-releasing factor and, recently, a group of peptides has been characterized from human pancreatic tumors (hpGRFs) (1,2) which are potent and specific GH-releasers in both animals (3) and man (4). The availability of these peptides, which show a high degree of homology with the physiologic rat hypothalamic GRF (5), offers the unique opportunity to assess somatotrope responsiveness to GRF molecules in rats with hypothalamo-pituitary disconnection.In this study we have first evaluated the GH pituitary responsiveness to increasing doses of hpGRF-40 in rats following mechanical ablation of the mediobasal hypothalamus (6). These rats, by definition, lack the effect of both central nervous system (CNS) inhibitory (e.g. somatostatin) and stimulatory (e.g. GRF) influences to GH release. With the aim to ascertain how the lack of these two opposing inputs reflects on the secretory capacity of the somatotropes, we also investigated the GH response to hpGRF-40 at different time intervals after the lesioning. In a study in rats with electrolytic lesions of the ventromedial-arcuate region of the hypothalamus Tannenbaum et al (7) had shown persistence of the GH response to huge doses of a hpGRF analog.     

Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

Background  

Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined.     

High-pass filtering of input signals by the Ih current in a non-spiking neuron, the retinal rod bipolar cell

Hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels mediate the I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of I(h) in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated-patch recordings in the dark-adapted slice found that RBCs exhibit I(h), and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency-modulated current stimuli (0.1-30 Hz), displays band-pass behavior in the range of I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), in combination with low-pass filtering by passive properties, fully accounts for this frequency-tuning. Correcting for the depolarization introduced by shunting through the pipette-membrane seal, leads to predict that in darkness I(h) is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1-2, in combination with markers of RBCs (PKC) and rod-RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.     

Genetic predictors of response to photodynamictherapy

In Western countries, therapeutic management of patients affected by choroidal neovascularization (CNV) secondary to different typologies of macular degeneration represents a major health care problem. Age-related macular degeneration is the disease most frequently associated with CNV development. Schematically, CNVs can be distinguished into classic and occult subtypes, which are characterized by variable natural history and different responsiveness to some therapeutic procedures. At present, the dramatic vision loss due to CNV can be mainly treated by two interventional strategies, which are utilizable in either single or combined modalities: photodynamic therapy with verteporfin (PDT-V), and intravitreal administration of drugs acting against vascular endothelial growth factor. The combined use of PDT-V and anti-angiogenic drugs represents one of the most promising strategies against neovascular macular degeneration, but it unavoidably results in an expensive increase in health resource utilization. However, the positive data from several studies serve as a basis for reconsidering the role of PDT-V, which has undergone a renaissance prompted by the need for a more rational therapeutic approach toward CNV. New pharmacogenetic knowledge of PDT-V points to exploratory prospects to optimize the clinical application of this intriguing photothrombotic procedure. In fact, a Medline search provides data regarding the role of several single nucleotide polymorphisms (SNPs) as genetic predictors of CNV responsiveness to PDT-V. Specifically, correlations between SNPs and different levels of PDT-V efficacy have been detected by examining the gene variants influencing (i) thrombo-coagulative pathways, i.e. methylenetetrahydrofolate reductase (MTHFR) 677C>T (rs1801133), factor V (F5) 1691G>A (rs6025), prothrombin (F2) 20210G>A (rs1799963), and factor XIII-A (F13A1) 185G>T (rs5985); (ii) complement activation and/or inflammatory processes, i.e. complement factor H (CFH) 1277T>C (rs1061170), high-temperature requirement factor A1 (HTRA1) promoter -512G>A (rs11200638), and two variants of the C-reactive protein (CRP) gene (rs2808635 and rs876538); and (iii) production and bioavailability of vascular endothelial growth factor (VEGFA -2578C>A [rs699947] and rs2146323). This article critically evaluates both the clinical plausibility and the opportunity to utilize the most important SNP-response interactions of PDT-V for an effective upgrade of the current anti-CNV therapeutic scenario. In addition, the pharmacogenetics of a very severe post-PDT-V adverse event, i.e. a decrease in acute vision, is briefly discussed. A comprehensive appraisal of the findings reviewed in this article should be carefully considered to design future trials aimed at verifying (after proper genotypic stratification of the enrolled patients) whether these innovative pharmacogenetic approaches will be able to improve the multifaceted interventional management of neovascular macular degeneration.     

Matric potential evaluations and measurements for gelled substrates

A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.     

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.