Pyrimidine Nucleotidases/Phosphotransferases from Human Erythrocyte

Abstract

Two cytoplasmic pyrimidine 5′-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5′-monophosphates and 3′-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5′-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.     

Growth stimulation of colorectal carcinoma cells via the c-kit receptor is inhibited by TGF-β1

Activation of the receptor tyrosine kinase c-kit by the kit-ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c-kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c-kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c-kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c-kit in situ. Expression of c-kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth-regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage-dependent and anchorage-independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)-β1 added at nanomolar concentrations to HT-29 CRC cells, which express the type I, II, and III TGF-β receptors, downregulated c-kit expression to background levels and inhibited c-kit–dependent proliferation. Similarly, TGF-β1 inhibited SCF-dependent proliferation of three first-passage CRC cell lines. In summary, expression of the potential autocrine SCF/c-kit axis is a tumor-associated phenomenon in colorectal cancer that can be suppressed by TGF-β1 in TGF-β–responsive CRC cells. J. Cell. Physiol. 172:1–11, 1997. © 1997 Wiley-Liss, Inc.     

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 μM), sodium nitroprusside (5 μM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1′-1′ Diphenyl-2′ picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

The role of p38α mitogen-activated protein kinase gene in the HELLP syndrome

Mitogen-activated protein kinase (MAPK) p38α was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38α in pregnancies complicated by HELLP. The placental expression of MAPK p38α was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38α is significantly decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38α is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38α. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.     

The effects of hypercaloric diets on glucose homeostasis in the rat: influence of saturated and monounsaturated dietary lipids

Consumption of energy-dense/high-fat diets is strongly and positively associated with overweight and obesity, which are associated with increase in the prevalence of certain chronic diseases. We evaluated the effect of hypercaloric/fat or normocaloric diets on some biochemical parameters in rats. Seventy-two rats were divided into four groups that were fed for 16 weeks with diets: normocaloric [9.12% soy oil, normocaloric soy oil (NSO)], hypercaloric olive oil [43.8% olive oil, hypercaloric olive oil (HOO)], hypercaloric saturated fat [43.8% saturated fat, hypercaloric saturated fat (HSF)] and normocaloric saturated fat [43.8% saturated fat, normocaloric saturated fat (NSF)]. HSF rats consumed more calories daily than the others and gained more retroperitoneal fat, although HSF and HOO rats had higher body weight. In liver, glycogen synthesis and concentration were higher in rats HSF and NSF. In plasma, total cholesterol (TC) levels were higher in HSF rats than in the others, and triacylglycerol (TAG) levels were lower in HOO and higher in HSF rats in relation to the others. In liver, TC and TAG were elevated in HSF, NSF and HOO rats. Paraoxonase 1 activity, which is related to high-density lipoprotein cholesterol and has anti-atherogenic role was lower in rats HSF. In HOO rats, glucose tolerance test was altered, but insulin tolerance test was normal. These results suggest that consumption of energy-dense/high-fat diets, both saturated or monounsaturated, causes damaging effects. However, more studies are necessary to understand the mechanisms by which these diets cause the metabolic alterations observed.     

Diterpene esters of aristolochic acids from Aristolochia pubescens

From the acetone and ethanol extracts of the tubercula of Aristolochia pubescens, two diterpene esters of aristolochic acids were isolated, together with 23 known compounds. The structures of the compounds were determined on the basis of spectroscopic analysis.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1-100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC50 was 1 nM. The response to oxytocin (0.01-10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desensitization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

Nuclear matrix-associated NMN adenylyltransferase activity in human placenta.

This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.