Crystal structures of a ferredoxin: NADP+ reductase and of a complex with NADP+.
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Anabaena↗
Biomedical subjects
M Medina
Publications and source records attributed to M Medina.
Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction.
Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule-associated protein termed tau, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of tau into filamentous structures, we have analyzed the ability of the whole tau protein or some of its fragments to self-assemble in the presence of heparin. Different tau fragments, all of them containing some sequences of the tubulin-binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18-residue-long peptide corresponding to the third tubulin-binding motif of tau. This suggests that the ability of tau for self-assembly could be localized in a short sequence of amino acids present in the tubulin-binding repeats of the tau molecule.
Actin Cytoskeleton↗
Cardiovascular effects of (-)-11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl in rats.
The effects of the stereochemically pure psychoactive cannabinoid (-)-11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl (HU-210) on blood pressure (BP) and heart rate (HR) were determined in rats. In pentobarbital-anesthetized animals, the compound produced dose-related, long-lasting hypotension and bradycardia at doses between 10 and 1,000 micrograms/kg. BP began to decrease immediately after drug administration, and in no case was an initial pressor response observed. Previous vagotomy (VX) or pretreatment with 6-hydroxydopamine (6-OHDA) did not affect hypotension. Bradycardia was inhibited by VX, but only 60 min after administration of HU-210; it was enhanced by 6-OHDA. The cannabinoid blocked reflex bradycardia induced by phenylephrine (PE). HU-210 also decreased BP and HR in conscious rats. Hypotension lasted 2 h, whereas bradycardia was still present 8 h after drug administration. HU-210 thus shares with delta 9-tetrahydrocannabinol (THC) the ability to decrease BP and HR, but is 5-10 times more potent than the natural compound. Its lack of an initial pressor effect, such as that described for THC, could be related to its specificity for the type-1 cannabinoid (CB1) receptor. Hypotension and bradycardia after HU-210 administration are not due to sympathetic withdrawal. Enhanced parasympathetic tone is involved in bradycardia only at a late stage of the response.
Animals↗
The epidermal growth factor receptor network in type 2 pneumocytes exposed to hyperoxia in vitro.
Hyperoxia is a well-characterized model of injury and repair of the lung. Type 1 cell damage is followed by type 2 cell proliferation and differentiation which restore normal structure and function. The epidermal growth factor receptor (EGFR) network is known to be a potent modulator of epithelial cell growth. Here we examine the EGFR network on isolated rat type 2 cells and SV40T-T2, a type 2 cell line, under normoxic conditions, after 24 and 48 h of in vitro hyperoxia, and after 24 h of normoxic recovery. EGF induces tyrosine phosphorylation of EGFRs in type 2 cells and SV40T-T2 cells, which decreases with hyperoxia and increases above normoxic levels in recovering cells, suggesting biphasic changes in receptor number or function with injury. The EGFR appears to be stimulated in an autocrine fashion in these cells. There is decreased DNA synthesis and proliferation in SV40T-T2 and isolated type 2 cells treated with tyrphostin B56, a specific EGFR inhibitor. Pretreatment with suramin, which binds to growth factor, results in increased EGFR tyrosine phosphorylation after stimulation, suggesting disruption of normal autocrine receptor downregulation. We have also identified transforming growth factor-alpha (TGF-alpha) in conditioned media (CM) from normoxic and hyperoxic SV40T-T2 and type 2 cells. Finally, we show increased EGF bioactivity in both bronchoalveolar lavage (BAL) from hyperoxic rats and CM from hyperoxic cells compared with normoxic controls. These findings support an integral role for an autocrine EGFR network in the type 2 cell response to injury.
Animals↗
Occurrence of Listeria monocytogenes and other Listeria spp. in raw caprine milk.
Samples of caprine milk from bulk tanks of 405 farms in central Spain were analyzed for Listeria spp. once per season over a 1-yr period. Listeria monocytogenes and Listeria innocua were detected in 2.56 and 1.73% of the 1445 samples, respectively. Listeria ivanovii (0.21% samples) and Listeria seeligeri (0.07% samples) were rarely isolated. Only 6 milk samples were contaminated by more than one Listeria species. Most farms (92.59%) produced milk that was apparently free from L. monocytogenes throughout the sampling period, and 88.40% produced milk that was apparently free from Listeria spp. Milk contamination by Listeria spp. was seasonal; incidence in autumn (9.33%) and winter (5.14%) samples was higher than incidence in spring (0.85%) and summer (0.85%) samples. Occurrence of Listeria spp. was lower in samples from mountain farms (1.25%) than in samples from plateau farms (7.03%).
Animals↗
Introduction of two new laparoscopic nooses.
Two devices for the temporary occlusion of tubular structures are introduced. They are constructed by combining a double-looped suture with a catheter segment. By this means, a terminal noose is created that can temporarily and atraumatically engage a vessel or other tubular structure. These devices can be used to obtain proximal and distal control of short segments without the need for bulky laparoscopic instruments in the operative field. The devices do not require purchase of any major equipment and can be made with any large-gauge polyfilament suture material and catheters available in the operating room.
Equipment Design↗
Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin).
Tau protein, the major component of the aberrant structures termed paired helical filaments (PHFs) present in the brain of Alzheimer's disease patients, is pathologically phosphorylated in sites in and around the tubulin-binding sites. A single protein kinase, glycogen synthase kinase 3 (GSK 3), is able to phosphorylate tau at the flanking regions and, additionally, at the tubulin-binding motifs if heparin or tubulin is present. Serines-262 and -324 have been found to be modified at the tubulin-binding region of tau protein by GSK 3 in the presence of heparin or tubulin.
Amino Acid Sequence↗
The role of tau phosphorylation in transfected COS-1 cells.
Tau cDNAs from each of the six human isoforms were transfected into COS-1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.
Actins↗
Involvement of nitric oxide in bone marrow-derived natural suppressor activity. Its dependence on IFN-gamma.
Bone marrow (BM)-derived natural suppressor (NS) cells are strong inhibitors of lymphoproliferative responses. In this study we have assessed the involvement of nitric oxide (NO) in BM-derived NS activity, as detected in cocultures of BM and spleen cells stimulated with B cell (LPS) or T cell (Con A) mitogens. The results indicate that NS activity is readily inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or N-acetylcysteine, a free radical-scavenging thiol compound. High amounts of nitrite, a stable end product of NO, are detected only in supernatants of Con A- or LPS-stimulated spleen cells cocultured with BM cells enriched in NS activity (Fr3 cells). These amounts (15 to 55 microM) are strongly antiproliferative for both Con A and LPS responses, as was established with a nitrite curve made with a NO donor (sodium nitroprusside). Fr3 cells cultured alone release large quantities of NO and express inducible NO synthase (iNOS) mRNA upon LPS stimulation, but require spleen cells in cultures stimulated with Con A. Anti-IFN-gamma-neutralizing Abs blocked both NO production and NS activity, irrespective of the mitogen used; yet, only exogenous IFN-gamma is unable to promote successful NO production by Fr3 cells, but does induce detectable iNOS mRNA expression in these cells. Taken together the results indicate that: 1) NO is the major mediator of BM-derived NS activity; 2) BM cells enriched in NS activity produce large amounts of NO through an IFN-gamma-dependent iNOS induction.
Acetylcysteine↗
Electron spin resonance and electron nuclear double resonance studies of flavoproteins involved in the photosynthetic electron transport in the cyanobacterium Anabaena sp. PCC 7119.
The flavins of ferredoxin-NADP+ reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H2O and 1.48 mT in D2O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP+, was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 7119 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in D2O, but the coupling Aiso attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP+. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D2O. Effects of NADP+ binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.(ABSTRACT TRUNCATED AT 400 WORDS)
Anabaena↗
[A methodology for the diagnosis and follow-up of the evolution of acute hepatitis in childhood. The Working Group of the Section of Pediatric Gastroenterology and Nutrition of the Portuguese Pediatrics Society].
The Working Group of the Section of Paediatric Gastroenterology and Nutrition of the Portuguese Society of Paediatrics established a protocol for the investigation of children with acute hepatitis. The main purpose of this proposal is to allow appropriate etiologic investigations and avoid unnecessary tests that are expensive and do not add relevant information for the correct follow-up of these patients.
Acute Disease↗
Introduction of a new double-looped suture.
Two new sutures are introduced for laparoscopic two-handed instrument suturing and knot tying, as well as a device for making them. In the first design, a nonslipping loop is placed at each end of a straight suture; they act as anchoring devices to maintain the suture in the two-dimensional plane of focus of the laparoscopic camera. The loops provide a large surface area that facilitates grasping the suture during the rotational and pulling maneuvers employed in instrument knot tying. The double-looped suture can be used for tying off and retracting tubular structures. The second design consists of a suture ligature with a curved or straight needle at one end and a single, non-slipping loop at the other. Illustrations demonstrate tying and suturing techniques for approximating tissue with the new sutures. A device consisting of two pairs of posts, for making the looped suture in a uniform fashion, is also discussed.
Humans↗
Identification of protein kinases that modify specific epitopes.
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Animals↗
Inhibition of bone marrow-derived natural suppressor activity by glucocorticoids and its reversal by IFN-gamma or IL-2.
Natural suppressor (NS) activity is mediated by cells (NS cells) of bone marrow origin with ability to suppress nonspecifically proliferative responses of lymphocytes. Here we show that pharmacologic concentrations (10(-6)-10(-8) M) of glucocorticoids (GC) greatly inhibit NS activity, as detected by coculturing bone marrow and spleen cells stimulated with B cell (LPS) or T cell (concanavalin A) mitogens. Progesterone antagonizes GC-mediated inhibition of NS activity, suggesting that GC were acting through a receptor-dependent mechanism. A prior treatment of NS cells with GC (10(-5) M) has no effect on the NS activity mediated by these cells. GC are required in culture during the first 24 hr of the suppressor assay. Addition of low amounts of IFN-gamma to GC-treated cultures fully reverses NS cell-mediated suppression. IL-2 produces a reversion as well, while addition of IL-3 or IL-4 does not prevent the GC effect. Neutralizing anti-IFN-gamma antibodies, but not anti-IL-2 or anti-TGF-beta, greatly inhibit NS activity in absence of GC. Taken together, these results indicate that GC inhibit NS activity by impairing endogenous cytokine production required to obtain successful NS cell activation, and not by acting directly on NS cells (i.e., inhibiting the secretion of putative NS factors). Among the cytokines involved in NS cell activation, IFN-gamma appears to be critical, since its addition readily overrides the GC effect and its neutralization results in strong inhibition of NS activity.
Animals↗
Further characterization by site-directed mutagenesis of the protein-protein interface in the ferredoxin/ferredoxin:NADP+ reductase system from Anabaena: requirement of a negative charge at position 94 in ferredoxin for rapid electron transfer.
Ferredoxins are small electron transfer proteins found ubiquitously in nature. In green plant photosynthesis, the soluble [2Fe-2S] ferredoxin shuttles electrons from Photosystem I to ferredoxin:NADP+ reductase. In order to define the features of the protein/protein interface required for efficient electron transfer from ferredoxin to ferredoxin:NADP+ reductase, we have made site-directed mutants of the ferredoxin from the cyanobacterium Anabaena 7120 and measured the rate constants for electron transfer to ferredoxin:NADP+ reductase using laser flash photolysis. Previous results from this laboratory identified two residues in ferredoxin that were crucial to electron transfer between these proteins. One such position (F65) was subsequently shown to require an aromatic amino acid, and it was further shown that interprotein electron transfer was rate-limiting in the case of the slowly reacting F65A mutant (Hurley et al., 1993 J. Am. Chem. Soc. 115, 11,698-11,701). The second crucial position (E94) is shown in the present study to require a negative charge in order to maintain wild-type-like electron transfer reactivity toward ferredoxin:NADP+ reductase. Further, we also demonstrate, for the slowly reacting E94Q mutant, that electron transfer is the rate-limiting step in the interprotein interaction.
Anabaena↗
ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state.
Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone. The rate and extent of reduction were pH-dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-1 cm-1. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 9-12 MHz, is attributed to protons on 8-CH3 (Aiso = 10.9 MHz) and on C6 (Aiso = 9 MHz) of the flavin ring. No change in these hyperfine coupling constants was detected when the protein was studied in D2O. However, the hyperfine coupling constant attributed to protons on 8-CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso = 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz. Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present. These signals are attributed to displaced water protons, or to exchangeable protons from amino-acid residues on the protein near the flavin binding site, involved in substrate stabilisation.
Binding Sites↗
Protein engineering for the elucidation of the mechanism of electron transfer in redox proteins.
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Anabaena↗
ESR and ENDOR characterization of flavodoxin from Anabaena sp. PCC 7119.
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Anabaena↗