Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.     

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.     

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.     

Role of chloride ions in modulation of the interaction between von Willebrand factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.     

New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards α-glucosidase

Several members of a new family of non-sugar-type α-glucosidase inhibitors, bearing a phthalimide moiety connected to a variously substituted phenoxy ring by an alkyl chain, were synthesized and their activities were investigated. The efficacy of the inhibition activity appeared to be governed by the chain length of the substrate. Substrates possessing 10 carbons afforded the highest levels of activity, which were one to two orders of magnitude more potent than the known inhibitor 1-deoxynojirimycin (dNM). Furthermore, structure–activity relationship studies indicated a critical role of electron-withdrawing substituents at the phenoxy group for the activity. Derivatives bearing a chlorine atom along with a strong electron-withdrawing group, such as a nitro group, were the most potent of the series.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Ochratoxin A adsorption phenotype: An inheritable yeast trait

This study aimed to evaluate the inheritance of the trait ochratoxin A adsorption in two wine strains of Saccharomyces cerevisiae and their 46 descendants. Each strain was inoculated in triplicate in test tubes containing 10 ml of must obtained from the Calabrian Zibibbo white grape variety, artificially contaminated with ochratoxin A to reach a total content of 4.10 ng/ml. The microvinification trials were performed at 25°C. After 30 days, ochratoxin A values ranged from 0.74 to 3.18 ng/ml, from 0.01 to 2.69 ng/ml, and from 0.60 to 2.95 ng/ml respectively in wines, in lees after washing, and in the saline solution used to wash the lees. The analysis of OTA in wines was performed to find the residual toxin content after yeast activity, thus obtaining technological evidence of yeast influence on wine detoxification. The analysis of OTA in lees after washing was performed to distinguish the OTA linked to cells. The analysis of OTA in the saline solution used to wash the lees was performed to distinguish the OTA adsorbed on yeast cell walls and removed by washing, thus focusing on the adsorption activity of wine yeast through electrostatic and ionic interactions between parietal mannoproteins and OTA. Ploidy of the two parental strains was controlled by flow cytometry. Results demonstrated that the ochratoxin A adsorption is genetically controlled and is a polygenic inheritable trait of wine yeasts. The majority of the descendants are characterized by a great and significant diversity compared to their parents. Both the parental strains had genome sizes consistent with their being diploid, so validating the observed results. These findings constitute an initial step to demonstrate the mechanisms of inheritance and establish breeding strategies to improve the ochratoxin A adsorption trait in wine yeasts. This will allow a decrease in the ochratoxin A content of contaminated musts during winemaking, by using genetically improved wine yeasts.