Structure elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 62D1.

The O-antigen polysaccharide of the lipopolysaccharide from the enteroaggregative Escherichia coli strain 62D1 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy revealed the components of the repeating unit. Two-dimensional NOESY and heteronuclear multiple-bond correlation experiments were used to deduce the sequence. 1H and 13C NMR spectra indicate heterogeneity in the polysaccharide. Methylation analysis and 1H NMR spectra of native and Smith-degraded material show that the majority (65%) of the repeating units has the following structure: Minor resonances in the NMR spectra are consistent with the presence of repeating units which lack the alpha-d-Galp terminal residue (35%).     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation

Summary.  Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [³H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced. Received March 15, 2001 Accepted July 8, 2002 Published online January 20, 2003 Acknowledgements This work was supported by the Grant S1-3490 from Consejo Nacional de Investigaciones Cientificas y Tecnológicas (CONICIT), Venezuela. We appreciate the secretarial assistance of Mrs. Isabel Otaegui. Carmen I. Gutiérrez is a PhD Student from Ciencias Fisiológicas, Facultad de Medicina, Universidad Central de Venezueta (UCV), Caracas, and supported by Universidad Francisco de Miranda, Coro, Falcón, Venezuela. Joseph Glykys is a Medical Student from Universidad de Carabobo, Valencia, Venezuela, and an Assistant Student of Centro de Estudios Avanzados, IVIC. Authors' address: Dr. Lucimey Lima, Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apdo. 21827, Caracas 1020-A, Venezuela, Fax: 58-212-504-1295, E-mail: llima@cbb.ivic.ve     

The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.     

Experimental Evidence for American Bullfrog (Lithobates catesbeianus) Susceptibility to Chytrid Fungus (Batrachochytrium dendrobatidis)

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been associated with global amphibian population declines and extinctions. American bullfrogs (Lithobates catesbeianus) are widely reported to be a tolerant host and a carrier of Bd that spreads the pathogen to less tolerant hosts. Here, we examined whether bullfrogs raised from eggs to metamorphosis in outdoor mesocosms were susceptible to Bd. We experimentally exposed metamorphic juveniles to Bd in the laboratory and compared mortality rates of pathogen-exposed animals to controls (non-exposed) in two separate experiments; one using a Bd strain isolated from a Western toad and another using a strain isolated from an American bullfrog. We wanted to examine whether metamorphic bullfrogs were susceptible to either of these strains. We show that bullfrogs were susceptible to one strain of Bd and not the other. In both experiments, infection load detected in the skin decreased over time, suggesting that metamorphic bullfrogs from some populations may be inefficient long-term carriers of Bd.     

Species sorting and mass effect along forest succession: Evidence from taxonomic,functional, and phylogenetic diversity of amphibian communities

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Delayed inflammation decrease is associated with mortality in Tocilizumab-treated critically ill SARS-CoV-2 patients: A retrospective matched-cohort analysis

Little is known about the immuno-inflammatory response to Tocilizumab and its association with outcome in critically-ill SARS-CoV2 pneumonia. In this multicenter retrospective cohort of SARS-CoV-2 patients admitted to three intensive care units between March and April 2020, we matched on gender and SAPS II 21 Tocilizumab-treated patients to 42 non-treated patients. Need for mechanical ventilation was 76% versus 79%. IL-6, C-reactive protein, and fibrinogen had been collected within the first days of admission (T1), 3 d (T2) and 7 d (T3) later. Tocilizumab-treated patients had persistently higher IL-6 plasma levels and persistently lower C-Reactive protein and fibrinogen levels. Among Tocilizumab-treated patients, baseline levels of inflammatory biomarkers were not different according to outcome. Conversely, C-reactive protein and fibrinogen decrease was delayed in non-survivors. C-Reactive protein decreased at T1 in survivors (45 [30–98] vs 170 [69–204] mg/l, P < 0.001) but only at T2 in non-survivors (37 [13–74] vs 277 [235–288], P = 0.03). Fibrinogen decreased at T2 in survivors (4.11 [3.58–4.69] vs 614 [5.61–7.85] g/l, P = 0.005) but not in non-survivors (4.79 [4.12–7.58] vs 7.24 [6.22–9.24] g/l, P = 0.125). Tocilizumab treatment was thus associated with a persistent both increase in plasma IL-6, and decrease in C-reactive protein and fibrinogen. Among Tocilizumab-treated patients, the decrease in inflammatory biomarkers was delayed in non-survivors.     

Crystal structure of IscS,a cysteine desulfurase from Escherichia coli

IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate-dependent desulfuration of L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. We report the crystal structure of Escherichia coli IscS to a resolution of 2.1A. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell dimensions a=73.70A, b=101.97A, c=108.62A (alpha=beta=gamma=90 degrees ). Molecular replacement with the Thermotoga maritima NifS model was used to determine phasing, and the IscS model was refined to an R=20.6% (R(free)=23.6%) with two molecules per asymmetric unit. The structure of E.coli IscS is similar to that of T.maritima NifS with nearly identical secondary structure and an overall backbone r.m.s. difference of 1.4A. However, in contrast to NifS a peptide segment containing the catalytic cysteine residue (Cys328) is partially ordered in the IscS structure. This segment of IscS (residues 323-335) forms a surface loop directed away from the active site pocket. Cys328 is positioned greater than 17A from the pyridoxal phosphate cofactor, suggesting that a large conformational change must occur during catalysis in order for Cys328 to participate in nucleophilic attack of a pyridoxal phosphate-bound cysteine substrate. Modeling suggests that rotation of this loop may allow movement of Cys328 to within approximately 3A of the pyridoxal phosphate cofactor.     

Specific chemotherapy of Chagas disease: controversies and advances

Chagas disease, caused by Trypanosoma cruzi, is a major public health problem in Latin America, where it constitutes one of the largest parasitic disease burdens. Specific treatment of this condition has been controversial, but there is a growing consensus that elimination of T. cruzi could be a prerequisite to arrest the evolution of the disease. Currently available chemotherapy, based on a nitrofuran (nifurtimox) and a nitroimidazole (benznidazole), is unsatisfactory because of their limited efficacy in the prevalent chronic stage of the disease and their toxic side effects. New approaches to specific chemotherapy are being advanced. Biochemical routes such as the de novo sterol biosynthesis pathway, cruzipain-mediated proteolysis and pyrophosphate metabolism have been chemically validated, and the selective in vitro and in vivo anti-T. cruzi activities of inhibitors of these pathways have been demonstrated. Several of these compounds have now completed pre-clinical studies and are poised for clinical trials in the near future. Other promising approaches include interference with trypanothione synthesis and redox metabolism, in addition to inhibition of purine salvage, dihydrofolate reductase, phospholipid biosynthesis, and protein prenylation and acylation.