Site-specific incorporation of the 1-hexanol-1,N6-etheno-2'-deoxyadenosine adduct into oligodeoxyribonucleotides.

Modified oligonucleotides that contain the hydrophobic 1-hexanol-1,N(6)-etheno-2'-deoxyadenosine adduct have been synthesized using a mild solid phase phosphoramidite chemistry. The presence and the integrity of the modified nucleoside in the synthetic oligomers were confirmed by electrospray ionization and MALDI mass spectrometry measurements together with analysis of the complete enzymatic hydrolysate by high performance liquid chromatography coupled to UV and fluorescent detection techniques.

Effect of in vivo administered hexachlorobenzene on epidermal growth factor receptor levels, protein tyrosine kinase activity, and phosphotyrosine content in rat liver.

In the present study, the effects of hexachlorobenzene (HCB) on epidermal growth factor receptor (EGFR) content of liver microsomes and plasma membrane, and on EGFR-tyrosine kinase activity in the microsomal fraction were investigated. In addition, we studied the parameters of the tyrosine kinase signalling pathway such as protein tyrosine kinase (PTK) activity and phosphotyrosine content in microsomal and cytosolic protein. To determine whether the observed alterations were correlated with a manifestation of overt toxicity, a single very low dose of HCB (1mg/kg body wt) and two much higher doses (100 and 1000 mg/kg body wt), the highest being toxicologically significant in that it reduced serum thyroxine (T(4)) and inhibited uroporphyrinogen decarboxylase (URO-D) (EC 4.

Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages.

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi.

Activity of isosteviol lactone on mitochondrial metabolism.

Isosteviol lactone (LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%), cytochrome c oxidase (10%), and NADH dehydrogenase (13%).

Memory extinction requires gene expression in rat hippocampus.

Rats with cannulae in the dorsal CA1 region of the hippocampus were trained in one-trial step-down inhibitory avoidance, and submitted to four consecutive daily test sessions without the footshock. This produced extinction of the conditioned response in control animals. The bilateral infusion into the CA1 region of the dorsal hippocampus of two different inhibitors of gene transcription, DRB (80 microg/side) or alpha-amanitin (25 pg/side), or of the protein synthesis inhibitor, anisomycin (80 microg/side) blocked extinction of the CR.

Alterations in the indexes of apoptosis and necrosis induced by galactosamine in the liver of Wistar rats treated with fructose-1,6-bisphosphate.

Galactosamine (GalN) is a hepatotoxic agent, which under determined situations provokes metabolic and energetic depletion as well as alterations in permeability, leading to cellular death. At the same time, it is known that fructose-1,6-bisphosphate (FBP) helps maintain cell energy levels and protects the cell against this lesive agent. We submitted two groups of male Wistar rats to the harmful intraperitoneal doses of GalN (400 mg/kg), one of which simultaneously received FBP (2 g/kg).

Hyperphenylalaninemia reduces creatine kinase activity in the cerebral cortex of rats.

Phenylketonuria (PKU) is a metabolic disorder accumulating phenylalanine (Phe) and its metabolites in plasma and tissues of the patients. Considering that phenylalanine is the main neurotoxic metabolite, and brain energy homeostasis seems to be affected in phenylketonuria, our main objective was to investigate the effect of acute and chronic hyperphenylalaninemia (HPA) on creatine kinase (CK) activity in brain cortex of Wistar rats. Hyperphenylalaninemia was induced by subcutaneous administration of 5.

Proline induces oxidative stress in cerebral cortex of rats.

In the present study we investigated the in vivo and in vitro effects of proline on some parameters of oxidative stress, such as chemiluminescence, total radical-trapping antioxidant potential (TRAP) and the activity of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in rat cerebral cortex. Ten-day-old rats received one subcutaneous injection of proline (12.8 micromol/g body weight), while control rats received saline in the same volumes.

Ligand-induced conformational changes of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves affecting the reactivity of the catalytic thiol.

The reaction catalyzed by betaine aldehyde dehydrogenase (BADH) involves the nucleophilic attack of a catalytic cysteinyl residue on the aldehyde substrate. As a possible mechanism of regulation, we have studied the modulation by ligands of the reactivity and/or accessibility of the essential thiol of the enzyme from the human pathogen Pseudomonas aeruginosa and the leaves of the plant Amaranthus hypochondriacus (amaranth). In the absence of ligands, the kinetics of inactivation by thiol modifying reagents of both enzymes were biphasic, suggesting the existence of two enzyme conformers differing in the reactivity of their catalytic thiolate.

Chemical denaturation of a homodimeric lysine-49 phospholipase A2: a stable dimer interface and a native monomeric intermediate.

Bothropstoxin I (4BthTx-I) is a homodimeric lysine-49 (Lys49) phospholipase A(2) isolated from Bothrops jararacussu venom, which damages liposome membranes via a Ca(2+)-independent mechanism. The stability of the BthTx-I homodimer was evaluated by equilibrium chemical denaturation with guanidinium hydrochloride monitored by changes in the intrinsic tryptophan fluorescence anisotropy, far-UV circular dichroism, dynamic light scattering, and 1-anilinonaphthalene-8-sulfonate binding. Unfolding of the BthTx-I dimer proceeds via a monomeric intermediate with native-like structure, with Gibbs free energy (DeltaG(0)) values of 10.

Mucin-type O-glycosylation in Fasciola hepatica: characterisation of carcinoma-associated Tn and sialyl-Tn antigens and evaluation of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity.

Simple mucin-type cancer-associated O-glycan structures, such as the Tn antigen (GalNAc-O-Ser/Thr), are expressed by certain helminth parasites. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. The aim of this work was to study the initiation pathway of mucin-type O-glycosylation in Fasciola hepatica, performing a biochemical and immunohistochemical characterisation of Tn and sialyl-Tn antigens, and evaluating the ppGaNTase activity, which catalyses the first step in O-glycan biosynthesis.

Oxidative damage to ferritin by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior.

5'-p-Fluorosulfonylbenzoyl adenosine inhibits progesterone synthesis in human placental mitochondria.

The human placental mitochondria have an ATP-diphosphohydrolase (apyrase) activity. In this paper we characterized the effect of 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) on placental apyrase, and its repercussion on progesterone synthesis and oxygen consumption. Apyrase activity was inhibited by FSBA.

The adenosine antagonist theophylline impairs p50 auditory sensory gating in normal subjects.

In the p50 suppression paradigm, when two auditory stimuli are presented 500 ms apart, the amplitude of the second response (S2), compared with the first (S1), is markedly attenuated in healthy subjects. This is an index of sensory gating. Most schizophrenic patients fail to inhibit the p50 response to the second stimulus, which is assumed to reflect an inhibitory deficit.

Chronic stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats.

We have previously observed that adenosine 5'-diphosphate (ADP) hydrolysis was decreased 25% in spinal cord synaptosomes of chronically stressed male rats, while no changes were observed in ATPase activity. In the present study, we investigated the effect of chronic stress on the hydrolysis of adenine nucleotides in two cerebral structures (frontal cortex and hypothalamus) and in the blood serum of male rats. Adult male Wistar rats were submitted to 1-h restraint stress/day for 45 days (chronic) and were sacrificed 24 h after the last session of stress.

Guanine and adenine nucleotidase activities in rat cerebrospinal fluid.

Adenine and guanine nucleotides have been shown to exert multiple roles in central and peripheral nervous systems, and the sequential breakdown of these nucleotides by enzymatic systems is an important step in the modulation of their extracellular effects. The aim of this study was to investigate whether nucleotide hydrolysis also occurs in the cerebrospinal fluid (CSF) of rats. CSF was able to hydrolyze all guanine and adenine nucleotides investigated (2.

The apolipoprotein B R3500Q gene mutation in Spanish subjects with a clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are autosomal codominant diseases characterized by elevated LDL cholesterol levels and premature coronary artery disease. Mutations of the LDL-receptor and apolipoprotein B genes, which affect the binding domains of their protein products, are the causal defects. Securing the diagnosis of these conditions by molecular assays is important because it mandates early intervention for coronary risk reduction.

Mayaro virus infection cycle relies on casein kinase 2 activity.

Replication of Mayaro virus in Vero cells induces dramatic cytopathic effects and cell death. In this study, we have evaluated the role of casein kinase 2 (CK2) during Mayaro virus infection cycle. We found that CK2 was activated during the initial stages of infection ( approximately 36% after 4h).

Ca(2+) modulates respiratory and steroidogenic activities of human term placental mitochondria.

We investigated the effects of calcium on the oxidative metabolism and steroidogenic activity of human term placental mitochondria. Submicromolar Ca(2+) concentrations stimulated state 3 oxygen consumption with 2-oxoglutarate and isocitrate and activated the 2-oxoglutarate and the NAD-isocitrate dehydrogenases by diminishing their Michaelis-Menten constants. Ca(2+) inhibited NADP-isocitrate dehydrogenase (NADP-ICDH) and the synthesis of progesterone.

Vampires, Pasteur and reactive oxygen species. Is the switch from aerobic to anaerobic metabolism a preventive antioxidant defence in blood-feeding parasites?

Several species of parasites show a reduction of their respiratory activity along their developmental cycles after they start to feed on vertebrate blood, relying on anaerobic degradation of carbohydrates to achieve their energy requirements. Usually, these parasites choose not to breathe despite of living in an environment of high oxygen availability such as vertebrate blood. Absence of the 'Pasteur effect' in most of these parasites has been well documented.

Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2.

Bothropstoxin-I (BthTx-I) is a Lys(49)-phospholipase A(2) from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca(2+)-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117-122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I.

Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state.

Fur (ferric uptake regulator) protein is a DNA-binding protein which regulates iron-responsive genes. Recombinant Fur from the nitrogen-fixing cyanobacterium Anabaena PCC 7119 has been purified and characterized, and polyclonal antibodies obtained. The experimental data show that Fur from Anabaena dimerizes in solution with the involvement of disulphide bridges.

4E binding protein 1 expression is inversely correlated to the progression of gastrointestinal cancers.

Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Overexpression of the initiation factor eIF4E occurs in a variety of human tumours. The aim of this study was to determine the level of expression and the phosphorylation state of eIF4E and 4E-binding protein 1 (4E-BP1) in gastrointestinal cancer, and to ascertain whether or not these factors can be used as diagnostic or prognostic markers within this type of cancer.

5-hydroxytryptamine1A receptor-mediated effects on adenylate cyclase and nitric oxide synthase activities in rat ventral prostate.

The rat ventral prostate possesses specific 5-hydroxytryptamine (5-HT1A) receptors coupled to adenylate cyclase. In vivo treatment of rats or in vitro preincubation of minced prostatic tissue with the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) in different experimental conditions shows the possibility of desensitisation mechanisms with switching from inhibitory to stimulatory pattern on adenylate cyclase activity. As in the majority of systems, we observed the inhibition of forskolin-stimulated adenylate cyclase activity as a functional correlate of 5-HT1A receptor activation.

Immune responses in Rhodnius prolixus: influence of nutrition and ecdysone.

Starved larvae of Rhodnius prolixus, when challenged with Enterobacter cloacae B12, had their mortality related to their period of starvation. R. prolixus larvae fed on plasma alone, compared with insects fed on whole blood, had their immune reactivity affected as shown by: (i) a significant reduction in the ability to produce cecropin-like and lysozyme activities in the haemolymph when inoculated with E.