Molecular dynamics and atomic charge calculations in the study of heparin conformation in aqueous solution

HF/6-31G** and molecular dynamics (MD) simulations were used to evaluate the performance of different atomic charge basis sets (i.e., Mulliken, Lowdin, and Electrostatic Potential Derived Charges--ESP) in heparin simulations. HF/3-21 G calculations were also used to study the NMR conformation of the IdoA residue. The results thus obtained indicated that ESP and Lowdin charges gave the better results in heparin simulations, followed by Mulliken charges, and that the minimum-energy conformation of IdoA can be different from that observed by NMR spectroscopy by less than 1 Angstrom. However, it was found that this small conformational modification is capable of inducing a change of almost 200 kJ/mol in the interactions of heparin with the surrounding environment, which is a meaningful amount of energy in the context of ligand-receptor interactions. This information can be potentially of great relevance in the design of heparin-derived antithrombotic compounds.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Intranigral LPS Administration Produces Dopamine,Glutathione but not Behavioral Impairment in Comparison to MPTP and 6-OHDA Neurotoxin Models of Parkinson’s Disease

The current investigation compared intranigral lipopolysaccharide (LPS), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) administrations, in the light of neurochemical, behavioral and endogenous antioxidant glutathione alterations. All the results were collected 1, 3 and 7 days after the lesions. LPS produced a delayed reduction of striatal dopamine, whereas homovanillic acid was drastically increased at the first time-point. Comparatively, MPTP promoted dopamine reduction 3 and 7 days with increase of homovanillic acid. Whilst, 6-OHDA generated initial increase of dopamine and homovanillic acid followed by subsequent decrease of this neurotransmitter accompanied by reductions of dopamine metabolites at the same periods. Furthermore, nigral glutathione demonstrated to be a far more sensitive target for LPS than for MPTP or 6-OHDA. Behavioral data indicated impairments induced by MPTP, 6-OHDA but not LPS. In conclusion, it is suggested that intranigral LPS can provide new insights about neuroinflammation, simulating features of the pre-motor phase of Parkinson’s disease.     

Evolution and population structure of Salmonella enterica serovar Newport

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.     

Paracoccin, a GlcNAc-binding lectin from Paracoccidioides brasiliensis, binds to laminin and induces TNF-alpha production by macrophages

Paracoccidioides brasiliensis components interact with host cells and can influence the pathogenesis of paracoccidioidomycosis (PCM). Among the components released by P. brasiliensis, gp 43 and a heavily glycosylated antigen with MM>160 kDa are the most recognized by serum antibodies from patients with PCM. In order to isolate the high MM glycoconjugate, we carried out affinity chromatography of a crude exoantigen preparation on immobilized jacalin. The bound fraction (JBE, jacalin binding exoantigen) consisted of a major antigen of high MM and frequently of an additional 70-kDa minor protein. This protein, designated paracoccin, exhibited selective binding to immobilized GlcNAc, a property that was used for its purification. The structural data of paracoccin obtained by mass spectrometry of tryptic peptides did not match any known protein. Anti-paracoccin serum localized the lectin on the surface of P. brasiliensis yeasts, especially in the budding regions. Paracoccin was able to interact with laminin in a dose-dependent manner. This interaction was inhibited by GlcNAc, followed by D-glucose and D-mannose, but not by D-galactose, N-acetyl-galactosamine or L-fucose. Interestingly, paracoccin induced both resident and elicited mouse peritoneal cavity macrophages to release high and persistent levels of TNF-alpha in vitro, a fact that was associated with high nitric oxide production in elicited cells. Because binding to laminin can favor yeast adhesion and invasion of host tissues, and overproduction of NO has been associated with suppression of cell immunity, paracoccin is suggested to play an important role in PCM pathogenesis.     

Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties

An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.     

Characterization of L-arginine transport in adrenal cells: effect of ACTH

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.     

Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB)

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.     

Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation

Hen egg white lysozyme (HEL), an antibacterial enzyme, is a prototype protein for studying the physical and chemical events that underlie the formation of amyloid fibril aggregates. Here, we studied alterations in enzymatic activity and aggregation provoked by oxidation of HEL by hypochlorous acid (HOCl), hypobromous acid (HOBr), taurine chloramine (Tau-NHCl), taurine monobromamine (Tau-NHBr), and taurine dibromamine (Tau-NBr(2)). Addition of only 4-fold molar excess of Tau-NHBr or Tau-NBr(2) to HEL caused complete depletion of its intrinsic fluorescence, whereas HOCl and HOBr caused 40%-50% bleaching. Tau-NHCl was unable to oxidize lysozyme. The selective effect of bromamines on tryptophan residues had a direct effect on enzymatic activity; bromamines were about two-fold more effective as inhibitors of lysozyme than the acid precursors. The oxidation of HEL by HOCl and HOBr was more effective regarding the aggregation of the protein, which was evidenced by increased turbidity, Rayleigh scattering, and anisotropy. The aggregates presented spectroscopic properties that suggested the formation of amyloid fibrils, as measured by the thioflavin assay. In conclusion, the capacity of Tau-NHBr and Tau-NBr(2) as inhibitors of the bactericidal activity of HEL could represent a role in the exacerbation of pulmonary infection, since leukocytes are rich sources of both taurine and HOBr. Moreover, the oxidation of HEL by just a small excess of hypohalous acids, a condition that could be found in inflammatory sites, may represent a new pathway for initiation of aggregation.