An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.     

Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.     

Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N‐terminal domain (DmI) of β2‐glycoprotein I (β2GpI). To possibly block anti‐β2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of β2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N‐DmI) was purified by RP‐HPLC, and its chemical identity and correct disulfide pairing (Cys4‐Cys47 and Cys32‐Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far‐ and near‐UV CD and fluorescence spectroscopy, provided strong evidence for the native‐like structure of DmI, which is also quite resistant to both Gdn‐HCl and thermal denaturation. However, the thermodynamic stability of N‐DmI at 37°C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N‐DmI in direct binding experiments. From ELISA competition experiments with plate‐immobilized β2GpI, a mean IC₅₀ value of 8.8 μM could be estimated for N‐DmI, similar to that of the full‐length protein, IC₅₀(β2GpI) = 6.4 μM, whereas the cysteine‐reduced and carboxamidomethylated DmI, RC‐DmI, failed to bind to anti‐β2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N‐terminally biotin‐(PEG)₂‐derivative of N‐DmI (Biotin‐N‐DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin‐N‐DmI loaded onto a streptavidin‐coated plate selectively recognized aAbs from APS patients.     

Pre-treatment of central venous catheters with the cathelicidin BMAP-28 enhances the efficacy of antistaphylococcal agents in the treatment of experimental catheter-related infection

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0x10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 microg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.     

Citropin 1.1-treated central venous catheters improve the efficacy of hydrophobic antibiotics in the treatment of experimental staphylococcal catheter-related infection

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of citropin 1.1, rifampin and minocycline. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with citropin 1.1 (10 microg/mL). Thirty minutes later the rats were challenged via the CVC with 1.0 x 10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC (the antibiotic lock technique) began 24 h later. The study included: one control group (no CVC infection), one contaminated group that did not receive any antibiotic prophylaxis, one contaminated group that received citropin 1.1-treated CVC, two contaminated groups that received citropin 1.1-treated CVC plus rifampin and minocycline at concentrations equal to MBCs for adherent cells and 1024 microg/mL in a volume of 0.1 mL that filled the CVC and two contaminated groups that received rifampin or minocycline at the same concentrations. All catheters were explanted 7 days after implantation. Main outcome measures were: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), synergy studies, quantitative culture of the biofilm formed on the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. MICs of conventional antibiotics against the bacteria in a biofilm were at least four-fold higher than against the freely growing planktonic cells. In contrast, when antibiotics were used on citropin 1.1 pre-treated cells they showed comparable activity against both biofilm and planktonic organisms. The in vivo studies show that when CVCs were pre-treated with citropin 1.1 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated both with citropin 1.1 and antibiotics, biofilm bacterial load was further reduced to 10(1) CFU/mL and bacteremia was not detected, suggesting 100% elimination of bacteremia and a log 6 reduction in biofilm load. Citropin 1.1 significantly reduces bacterial load and enhances the effect of hydrophobic antibiotics in the treatment of CVC-associated S. aureus infections.     

Ultrastructural and cytochemical aspects of the female gonad of Geoplana burmeisteri (Platyhelminthes, Tricladida, Terricola)

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.