Antioxidant mechanisms of the Nereidid Laeonereis acuta (Anelida: Polychaeta) to cope with environmental hydrogen peroxide

Hydrogen peroxide (H(2)O(2)) is a naturally occurring prooxidant molecule, and its effects in the macroinvertebrate infauna were previously observed. The existence of a gradient of antioxidant enzymes activity (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD], and glutathione-S-transferase [GST]) and/or oxidative damage along the body of the estuarine polychaeta Laeonereis acuta (Polychaeta, Nereididae) was analyzed after exposure to H(2)O(2). Because this species secretes conspicuous amounts of mucus, its capability in degrading H(2)O(2) was studied. The results suggest that L. acuta deal with the generation of oxidative stress with different strategies along the body. In the posterior region, higher CAT and SOD activities ensure the degradation of inductors of lipid peroxidation such as H(2)O(2) and superoxide anion (O(2)(.-)). The higher GST activity in anterior region aids to conjugate lipid peroxides products. In the middle region, the lack of high CAT, SOD, or GST activities correlates with the higher lipid hydroperoxide levels found after H(2)O(2) exposure. Ten days of exposure to H(2)O(2) also induced oxidative stress (lipid peroxidation and DNA damage) in the whole animal paralleled by a lack of CAT induction. The mucus production contributes substantially to H(2)O(2) degradation, suggesting that bacteria that grow in this secretion provide this capability.     

Dry granulation and compression of spray-dried plant extracts

The purpose of this research was to evaluate the influence of dry granulation parameters on granule and tablet properties of spray-dried extract (SDE) fromMaytenus ilicifolia, which is widely used in Brazil in the treatment of gastric disorders. The compressional behavior of the SDE and granules of the SDE was characterized by Heckel plots. The tablet properties of powders, granules, and formulations containing a high extract dose were compared. The SDE was blended with 2% magnesium stearate and 1% colloidal silicon dioxide and compacted to produce granules after slugging or roll compaction. The influences of the granulation process and the roll compaction force on the technological properties of the granules were studied. The flowability and density of spray-dried particles were improved after granulation. Tablets produced by direct compression of granules showed lower crushing strength than the ones obtained from nongranulated material. The compressional analysis by Heckel plots revealed that the SDE undergoes plastic deformation with a very low tendency to rearrangement at an early stage of compression. On the other hand, the granules showed an intensive rearrangement as a consequence of fragmentation and rebounding. However, when the compaction pressure was increased, the granules showed plastic deformation. The mean yield pressure values showed that both granulation techniques and the roll compaction force were able to reduce the material's ability to undergo plastic deformation. Finally, the tablet containing a high dose of granules showed a close dependence between crushing strength and the densification degree of the granules (ie, roll compaction force). Published: October 14, 2005     

Chromatographic evaluation of the toxicity in fish of pesticides

Ecotoxicity assessment is essential before placing new chemical substances on the market. An investigation of the use of the chromatographic retention (log k) in biopartitioning micellar chromatography (BMC) as an in vitro approach to evaluate the toxicity in fish of pesticides (acute toxicity levels as pLC(50)) is proposed. A heterogeneous data set of 85 pesticides from six chemical families with available experimental fish toxicity data (ECOTOX database from U.S. Environmental Protection Agency (EPA)) was used. For pesticides exhibiting non-polar narcosis mechanism in fish (non-specific toxicity), more reliable models and precise pLC(50) estimations are obtained from log k (quantitative retention-activity relationships, QRAR) than from log P (quantitative structure-activity relationships, QSAR) or ECOSAR (ECOSAR program from U.S. EPA).     

Candida riodocensis and Candida cellae, two new yeast species from the Starmerella clade associated with solitary bees in the Atlantic rain forest of Brazil

Two new ascomycetous yeast species belonging to the Starmerella clade were discovered in nests of two solitary bee species in the Atlantic rain forest of Brazil. Candida riodocensis was isolated from pollen-nectar provisions, larvae and fecal pellets of nests of Megachile sp., and Candida cellae was found in pollen-nectar provisions of Centris tarsata. Analysis of the sequences of the D1/D2 large-subunit ribosomal DNA showed that C. riodocensis is phylogenetically related to C. batistae, and the closest relative of C. cellae is C. etchellsii. The type strains are C. riodocensis UFMG-MG02 (=CBS 10087(T) = NRRL Y-27859(T)) and C. cellae UFMG-PC04 (=CBS 10086(T) = NRRL Y-27860(T)).     

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus

Clavulanic acid (CA), a potent β-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O₂ (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O₂, were performed. Significant variations in CA titer, CA production rate and O₂ uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.     

The burgeoning molecular genetics of the Lyme disease spirochaete

Lyme disease is the most commonly reported vector-borne disease in North America and Europe, yet we know little about which components of the causative agent, Borrelia burgdorferi, are critical for infection or virulence. Molecular genetics has provided a powerful means by which to address these topics in other bacterial pathogens. Certain features of B. burgdorferi have hampered the development of an effective system of genetic analysis, but basic tools are now available and their application has begun to provide information about the identities and roles of key bacterial components in both the tick vector and the mammalian host. Increased genetic analysis of B. burgdorferi should advance our understanding of the infectious cycle and the pathogenesis of Lyme disease.     

Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.     

Evidence for a granule targeting sequence within platelet factor 4

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed alpha-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory alpha-granule chemokine, and demonstrated that PF4 alpha-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 +/- 2% to 17 +/- 3%, p < or = 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in alpha-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 +/- 1.8% colocalization with the alpha-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human alpha-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same alpha-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to alpha-granule targeting of chemokines and possibly of other alpha-granule proteins.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.