Souvenir Tachado - Academia.edu (original) (raw)

Papers by Souvenir Tachado

Indian journal of biochemistry & biophysics

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in th... more We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

A: Small RNAs processed from HIV-1 LTR region observed by SOLiD Deep ... more A: Small RNAs processed from HIV-1 LTR region observed by SOLiD Deep Sequencing. Left peak shows small RNAs derived from TAR stem (miR TAR). Right peak shows a hotspot for small RNAs derived from R and U5 stem region. The GU-rich tract (46 nt) encodes a family of viral miRs including vmiR88 and vmiR99. Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Schopman1" target="_blank">[14]</a>. B: shRNA mirs are intermediates in biogenesis of mature vmiRs. shRNA reported for 43/9175 TAR (left). UNAFold software predicts folding of shRNAs vmir88 (middle) and vmir99 (right), which suggests the structures of intermediates in the biogenesis of the mature vmiR-TAR (black rectangle), vmiR88 (blue rectangle) and vmiR99 (red rectangle). UNAFold's thermodynamic calculations predict that all three shRNAs fold spontaneously (ΔG<0) into stable hairpins (high melting temperature, TM>53.8°C in 1M Na+). C: To delineate the boundaries and sequences of mature viral miRNAs, cell extracts and exosomal extracts were analyzed. Sample cell extracts were in vitro-infected AM (healthy AM+HIV), HIV-positive U1 macrophages stimulated by PMA (U1+PMA). Exosomal extracts were from exosomes of HIV+ human serum (HIV+ serum 10 b). Total RNA was amplified by qRT-PCR, cloned into pCR4-TOPO vector and DNA was sequenced. Sequences of vmiR88 and vmiR99 PCR products were aligned with sequences of plasmid (vector) and HIV-BaL strain. The polyadenylation signal (PA signal) and polyadenylation site (PA site) were reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Bhnlein1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Berkhout1" target="_blank">[36]</a>.</p

Purpose: To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can ind... more Purpose: To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. Methods: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFa by macrophages challenged with HIV miRNAs was measured by ELISA. Results: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFa release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs relea...

Cell Signal, 1992

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluo... more We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.

Journal of Cellular Physiology, 2007

European Journal of Pharmacology Molecular Pharmacology Section, Aug 3, 1992

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulati... more In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscar]nic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mamtp.alian species were found t,9 be coupled to phospholipase C and contraction at all concentrations of earbachol investigated (1-100/z M). In the dog sphincter, lower concentrations (< 5 #M) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP0 production, inhibited cAMP formation and induced contraction, and higher concentrations (> 5/aM) enhanced cAMP formation, inhibited IP 3 production and induced re!a×ation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25 tJ.M) increased both basal and isoprotel'enot-and forskoiit~-stin~ulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC5( ~ of 9 nM. lntraceilular Ca"+, derived from !P3-induced Ca -'+ release and/or from muscarinic receptor-operated Ca 2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (1) At short time intervals ( < 1 min) carbacho! (25 t~M) increased IP 3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated 1P 3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbot ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca2+-calmodulin stimulated adenylate cyclase was demonstrated in mcmb~anes fr~,m dog iris sphincter but not in that from rabbit and bovine. (,~) Trifluoperazine (C.I ~M)~ a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (51 The Ca z+ ionophore ATq187 ~tnd the phorbol ester increased cAM):' production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carb~cho[ or by 'he phnrbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol e~,:er or staurosporine. (7) Nifedipine (0.01-0.5/aM), a Ca 2+ channel antagonist, inhibited carbachol stimulation of cAMP pr(~daction, suggesting the presence of a muscarinic receptor-operated Ca z+ influx pathway in this tissue. The above data are interesting in three ways: (a) They demonstrate an important species difference in the second messenger systems coupled to the activation of muscarin:c receptors, (.O) they demonstrate a reciprocal re!ationsifip (c~o~s-ta|k) bctwcc~ cAMP af~d IP~ pl~oduction, and (c) they suggest that both intracellular and cxtracellular Ca -'+ mobilization may be involved in muscarinic sti~nulation of cAMP production. We suggest that in dog iris sphincter a rise in intraeeltular cAMP levels by cholinergic stimulation is needed to regulate muscarinic receptor-linked Ca -'+ mobilization and mu~le contraction-relaxation. Cyclic AMP production through muscarinic activation could also be required for the functioning of Ca '~ channels in this excitable tissue.

American Journal of Respiratory Cell and Molecular Biology, Dec 20, 2012

ABSTRACT

The Journal of Biological Chemistry, 2008

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge... more Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.

Investigative Ophthalmology &amp Visual Science

(1) Addition of 5 nM isoproterenol (ISO) or forskolin (5 yM) consistently produced stimulation of... more (1) Addition of 5 nM isoproterenol (ISO) or forskolin (5 yM) consistently produced stimulation of cAMP (540%), inhibition of IP 3 (34%) and complete relaxation of the muscle. The ISO effects were dose-dependent, with ECso values for cAMP formation, IP 3 inhibition and muscle relaxation of 2.8 X 10~7 M, 3.4 X 10~7 M and 0.45 X 10" 7 M, respectively. (2) Timolol, a /?-adrenergic antagonist, inhibited the ISO effects in a dose-dependent manner, with IC50 values for cAMP formation, IP 3 accumulation and muscle relaxation of 1.8 X 10~5 M, 3.2 X 10" s M and 2.2 X 10" 5 M, respectively. (3) The effects of ISO (0.5 nM) were time-dependent, and they clearly indicate a temporal relationship between the agonist-induced stimulation of cAMP, inhibition of IP 3 , and relaxation of the muscle. Within 15 sec following the addition of ISO, there was a marked increase in the level of cAMP, a decrease in IP 3 , and this was accompanied by an equally rapid relaxation of the muscle. (4) Addition of carbachol (CCh) to iris sphincter pretreated with ISO decreased cAMP formation and reversed muscle relaxation to complete contraction. Thus, when the sphincter was first treated with ISO and then stimulated with different concentrations of CCh, there was a dose-dependent inhibition of cAMP formation, an increased production of IP 3 , and a parallel development of muscle contraction. Taken together, the data presented suggest a reciprocal interaction between the cAMP and IP 3 -Ca 2+ signalling systems in the iris sphincter: activation of /?-adrenergic receptors results in elevation of cAMP and inhibition of IP 3 , and this leads to muscle relaxation, whereas stimulation of cholinergic muscarinic receptors lowers cAMP formation and increases IP 3 production, and this leads to Ca 2+ mobilization and muscle contraction. We propose that in the iris sphincter cAMP may act as regulator of responses to neurotransmitters, hormones and pharmacological agents that exert their action through the IP 3 -Ca 2+ second messenger system. Invest Ophthalmol Vis Sci

Current Eye Research

The effect of short-term prostaglandin (PG) desensitization on PGF2 alpha receptor-mediated inosi... more The effect of short-term prostaglandin (PG) desensitization on PGF2 alpha receptor-mediated inositol phosphates accumulation, 1,2-diacylglycerol production, measured as phosphatidic acid (PA), myosin light chain (MLC) phosphorylation, cAMP formation and contraction was investigated in bovine iris sphincter smooth muscle. We have found that incubation of the sphincter with 25 microM PGF2 alpha for 45 min leads to: (a) significant loss in sensitivity of the tissue to PGF2 alpha receptor-stimulated inositol phosphates accumulation, PA production, MLC phosphorylation and contraction, and (b) significant increase in both basal and PGF2 alpha-stimulated cAMP formation. These changes are probably not due to reduction in phospholipid synthesis because there were no detectable differences in basal phospholipid labeling, either from 3H-inositol or from 32P, between normal and desensitized muscles. Preincubation of the sphincter in the absence of PGF2 alpha for 45 min did not lead to alterations in the biochemical-pharmacological responsiveness of the control muscle to PGF2 alpha. Our results suggest that desensitization of PG receptors in the iris sphincter occurs by a receptor-specific process. The PG receptor mediating contraction (IP3-Ca2+) is selectively susceptible to desensitization, in contrast with the receptor mediating smooth muscle relaxation (cAMP). These findings add further support to the developing hypothesis that there are functional and biochemical reciprocal interactions between the IP3-Ca2+ and cAMP messenger systems in the iris of the mammalian eye.

The Journal of Immunology

ABSTRACT

The Journal of Immunology

In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmod... more In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum origin responsible for nitric oxide (NO) production in host cells. Purified malarial GPI is sufficient to induce NO release in a time- and dose-dependent manner in macrophages and vascular endothelial cells, and regulates inducible NO synthase expression in macrophages. GPI-induced NO production was blocked by the NO synthase-specific inhibitor L-N-monomethylarginine. GPI also synergizes with IFN-gamma in regulating NO production. The structurally related molecules dipalmitoylphosphatidylinositol and iM4 glycoinositolphospholipid from Leishmania mexicana had no such activity, and the latter antagonized IFN-gamma-induced NO output. GPI activates macrophages by initiating an early onset tyrosine kinase-mediated signaling process, similar to that induced by total parasite extracts. The tyrosine kinase antagonists tyrphostin and genistein inhibited the release of NO by parasite extracts and by GPI, alone or in combination with IFN-gamma, demonstrating the involvement of one or more tyrosine kinases in the signaling cascade. GPI-induced NO release was also blocked by the protein kinase C inhibitor calphostin C, demonstrating a role for protein kinase C in GPI-mediated cell signaling, and by pyrrolidine dithiocarbamate, indicating the involvement of the NF-kappa B/c-rel family of transcription factors in cell activation. A neutralizing mAb to malarial GPI inhibited NO production induced by GPI and total malarial parasite extracts in human vascular endothelial cells and murine macrophages, indicating that GPI is a necessary agent of parasite origin in parasite-induced NO output. Thus, in contrast to dipalmitoylphosphatidylinositol and glycoinositolphospholipids of Leishmania, malarial GPI initiates a protein tyrosine kinase- and protein kinase C-mediated signal transduction pathway, regulating inducible NO synthase expression with the participation of NF-kappa B/c-rel, which leads to macrophage and vascular endothelial cell activation and downstream production of NO. These events may play a role in the etiology of severe malaria.

Investigative Ophthalmology &amp Visual Science

To determine if there is endothelin-mediated regulation of cell signaling and proliferation in ra... more To determine if there is endothelin-mediated regulation of cell signaling and proliferation in rabbit corneal epithelium. Endothelin-1 (ET-1) gene and protein expression by the rabbit corneal epithelial (RCE) cells were analyzed by polymerase chain reaction, sequence analysis, and enzyme immunoassay. DNA synthesis was characterized by [3H]-thymidine uptake. Endothelin receptor linkage to cell signaling pathways was determined based on measurements of the dose dependent effects of ET-1, ET-2, and ET-3 on intracellular Ca2+ concentration ([Ca2+]i) transients in fura-2-loaded cells, and of ET-1 on phosphoinositide turnover and cAMP accumulation in the isolated rabbit corneal epithelium. The authors detected the mRNA for prepro ET-1 in RCE cells, and ET-like immunoreactivity was identified in conditioned culture medium. ET-1 (1 nM) maximally stimulated [3H]-thymidine uptake by twofold (EC50 = 0.3 nM). Endothelins elicited transient increases in [Ca2+]i with a rank order of potency of ET-1 > or = ET-2 > ET-3. These increases consisted of both intracellular Ca2+ mobilization and influx of Ca2+ from the bathing solution. Intracellular mobilization was linked to increases in IP3 turnover because 1 microM ET-1 increased IP3 content by 48% from its control value (EC50 = 23 nM), whereas Ca2+ influx occurred through a non-L-type Ca2+ channel because preexposure to 1 microM nicardipine did not affect either the height or the duration of a [Ca2+]i transient. One micromolar of ET-1 was required to elicit a significant increase in cAMP accumulation of 69% from its control value. This increase was dependent on the presence of Ca2+ in the bathing solution and was comparable to and nonadditive with that of the Ca2+ ionophore, A23187 (1 microM). These data suggest that endothelin production by primary cultures of RCE cells can mediate an increase in cell proliferation through an ETA receptor subtype. This receptor subtype appears to be involved based on the rank order of potency of ETs to elicit [Ca2+]i transients, increases in phosphoinositide turnover, and cAMP accumulation.

Brazilian Journal of Medical and Biological Research

Considerable circumstantial evidence indicates that glycosylphosphatidylinositol (GPI) molecules ... more Considerable circumstantial evidence indicates that glycosylphosphatidylinositol (GPI) molecules of mammalian origin are able to mediate signal transduction in lymphoid cells. For example, perturbation of GPI-anchored surface proteins, but not transmembrane forms of these molecules, can lead to the activation of T lymphocytes. GPIs appear also to be precursors of pharmacologically active phosphoinositol-glycans which mediate responses to hormones such as insulin, nerve growth factor and IL-2. Nonetheless, the biochemical mechanisms of signal transduction by GPIs remain obscure. We have shown that structurally defined GPIs of protozoal parasite origin are able to mediate signal transduction in host macrophages and lymphocytes, by substituting for the putative endogenous GPI-based signalling mechanisms of the host. Signalling by parasite GPIs appears to involve the activation of protein tyrosine kinase and protein kinase C. Evidence from other sources indicates that structurally variant GPIs may provide anergic signals to down-regulate host cell function. These phenomena may represent mechanisms by which eukaryotic parasites regulate host cell function, and can explain a variety of pathological and immunological features of protozoal infections. Furthermore, protozoal GPIs may prove to be an informative model system for the analysis of GPI-mediated signal transduction in lymphocytes and macrophages.

The molecular mechanisms for increased risk of bacterial pneumo- nia in HIV persons remain incomp... more The molecular mechanisms for increased risk of bacterial pneumo- nia in HIV persons remain incompletely understood. Recognizing the critical role of Toll-like receptor (TLR) signaling in host defense, this study showed that human U937 macrophage stimulation by the TLR4-specific ligand, lipid A (biologically active component of bacterial LPS), promoted TNF- release through extracellular regu- lated kinase (ERK)1/2 mitogen-activated protein (MAP)

Molecular Medicine Reports, 2015

Prostate cancer (PCa) is the second leading cause of cancer-related mortality among American male... more Prostate cancer (PCa) is the second leading cause of cancer-related mortality among American males. Studies suggest that cigarette smoking is associated with the progression of PCa; however, the molecular mechanisms underlying this process have not been extensively investigated. PCa progression is characterized by increased cell migration and alterations in extracellular matrix (ECM)-and cell adhesion molecule (CAM)-related gene expression. In the present study, the influence of cigarette smoke medium (SM) on cell migration and on the expression of ECM-and CAM-related genes in PC3 prostate adenocarcinoma cells was investigated. According to a wound-healing assay, SM treatment promoted PC3 cell migration. RNA expression levels from SM-treated and control cells were analyzed using a polymerase chain reaction (PCR) array. Of 84 genes analyzed, 27.38% (23/84) exhibited a ≥2-fold change in threshold cycle in PC3 cells following 0.5% SM treatment. Functional gene grouping analysis demonstrated that SM treatment modulated the RNA transcription of approximately 18.4% of CAMs and 33.93% of ECM-related genes. Quantitative PCR analysis showed that SM treatment led to a significant decrease in transcription levels of the following genes: Collagen 5 α-1(V), connective tissue growth factor, integrin β-2, kallmann syndrome 1, laminin α 3, matrix metallopeptidase 7 (MMP7), MMP13, secreted protein acidic cysteine-rich, thrombospondin-2 and versican; and that SM significantly increased the transcription levels of MMP2 and MMP12. Furthermore, MMP2 knockdown significantly reduced the migration of SM-treated PC3 cells. The present study provides novel insights into the association of cigarette smoking with PCa progression, via the alteration of ECM/CAM interactions.

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MT... more The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-.

Background: Chronic immune activation may be in part attributable to translocation of microbial T... more Background: Chronic immune activation may be in part attributable to translocation of microbial TLR ligands from the GI tract into the systemic circulation, but may involve direct effects of viral proteins and nucleic acids and activation of bystander host immune cells. We investigated whether novel HIV-derived miRNAs can directly induce macrophage proinflammatory cytokine release via non-canonical pathway. Methodology: HIV miRNAs including vmiRNA-TAR and two novel HIV miRNA designated as vmiR88 and vmiR99 (determined by UNAfold RNA folding software, from R/U5 and U5 regions of HIV LTR, each with requisite short hairpin structure and highly conserved sequence vs. several clinical HIV isolates) were synthesized by IDT (Coralville, IA). Human macrophages U937 and HIV+U1 cell lines (differentiated with PMA), and human alveolar macrophages (AM) from consenting healthy or asymptomatic HIV+ volunteers (hospital IRB-approved bronchoscopy protocol) were isolated by adherence. For select exp...

Indian journal of biochemistry & biophysics

Cell Signal, 1992

Journal of Cellular Physiology, 2007

European Journal of Pharmacology Molecular Pharmacology Section, Aug 3, 1992

American Journal of Respiratory Cell and Molecular Biology, Dec 20, 2012

ABSTRACT

The Journal of Biological Chemistry, 2008

Investigative Ophthalmology &amp Visual Science

Current Eye Research

The Journal of Immunology

ABSTRACT

The Journal of Immunology

Investigative Ophthalmology &amp Visual Science

Brazilian Journal of Medical and Biological Research

Molecular Medicine Reports, 2015