The lipid-mobilizing effect of atrial natriuretic peptide is unrelated to sympathetic nervous system activation or obesity in young men

We recently demonstrated that natriuretic peptides and especially the atrial natriuretic peptide (ANP) are powerful lipolytic agents on isolated human fat cells. To search for a possible influence of obesity on ANP responsiveness, we compared the lipolytic effects of human ANP (h-ANP) on isolated subcutaneous abdominal adipose tissue (SCAAT) fat cells from young healthy lean and obese men. The lipid-mobilizing effects of an intravenous infusion of h-ANP was studied, as well as various metabolic and cardiovascular parameters that were compared in the same subjects. h-ANP (50 ng/min/kg) was infused iv for 60 min. Microdialysis probes were inserted in SCAAT to measure modifications of the extracellular glycerol concentrations during h-ANP infusion. Spectral analysis of blood pressure and heart rate oscillations that were recorded using digital photoplethysmography were used to assess changes in autonomic nervous system activity. h-ANP induced a marked and similar increase in glycerol and nonesterified fatty acids, and a weak increase in insulin plasma levels in lean and obese men. Plasma norepinephrine concentrations rose similarly during h-ANP infusion in lean and obese men. The effects of h-ANP infusion on the autonomic nervous system were similar in both groups, with an increase in the spectral energy of the low-frequency band of systolic blood pressure variability and a decrease in the spectral energy of the high-frequency band of heart rate. In SCAAT, h-ANP infusion increased extracellular glycerol concentration and decreased blood flow similarly in both groups. The increase in extracellular glycerol observed during h-ANP infusion was not modified when 0.1 mM propranolol was added to the microdialysis probe perfusate to prevent beta-adrenoceptor activation. These data show that ANP is a potent lipolytic hormone independent of the activation of the sympathetic nervous system, and that obesity did not modify the lipid-mobilizing effect of ANP in young obese subjects.     

Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.     

Distribution of glycogen phosphorylase and cytochrome oxidase in the central nervous system of the turtle Trachemys dorbigni

Glycogen phosphorylase (GP) and cytochrome oxidase (CO) activities were mapped histochemically in the brain of the turtle Trachemys dorbigni. In the telencephalon, both activities occurred in the olfactory bulb, in all cortical areas, in the dorsal ventricular ridge, striatum, primordium hippocampi and olfactory tubercle. In the diencephalon, they were identified in some areas of the hypothalamus, and in rotundus and geniculate nuclei. Both reactions were detected in the oculomotor, trochlear, mesencephalic trigeminal nuclei, the nucleus of the posterior commissure, torus semicircularis, substantia nigra and ruber and isthmic nuclei of the mesencephalon. In all layers of the optic tectum GP activity was found, but CO only labelled the stratum griseum centrale. In the medulla oblonga both enzymes appear in the reticular, raphe and vestibular nuclei, locus coeruleus and nuclei of cranial nerves. In the cerebellum, the granular and molecular layers, and the deep cerebellar nuclei were positive for both enzymes. The Purkinje cells were only reactive for CO. In the spinal cord, motor and commissural neurones exhibited a positive reaction for the two enzymes. However, CO also occurred in the marginal nucleus and in the lateral funiculus. These results may be useful as a basis for subsequent studies on turtle brain metabolism.     

Homophilic NCAM interactions interfere with L1 stimulated neurite outgrowth

The cell adhesion molecules NCAM and L1 are considered to play key roles in neuronal development and plasticity. L1 has been shown to interact with NCAM, possibly through NCAM binding to oligomannosidic glycans present in L1. We investigated the effect of recombinant immunoglobulin (Ig) modules of NCAM involved in homophilic NCAM binding, on L1 induced neurite outgrowth from PC12-E2 cells and found a complete inhibition of L1 induced neurite outgrowth after addition of Ig-modules 1, 2 and 3 of NCAM, suggesting that the ligation state of NCAM is crucial for normal L1 signaling.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin--an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe(2)(CO(3))(2)BTf], [Fe(2)(C(2)O(4))(2)BTf], [Cu(2)(CO(3))(2)BTf] and [Cu(C(2)O(4))BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal-oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C(2)O(4))BTf], the mixed-anion complexes [Cu(2)(CO(3))(C(2)O(4))BTf] and [Fe(2)(CO(3))(C(2)O(4))BTf] and the mixed-metal complex [FeCu(C(2)O(4))(2)BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p(pi)-d(pi*) CT band for iron(III)-BTf and the hypsochromic shift of the p(pi)-d(sigma*) CT band for copper(II)-BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin--a serum transferrin--very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.     

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH₂ (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH₂ (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the -amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.     

Continued proteomic analysis of Mycobacterium leprae subcellular fractions

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.     

Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37°C and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.Communicated by C P. Hollenberg     

Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid

Several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. The new heterocycles were evaluated for their activity in vitro against several RNA and DNA viruses.