A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase

Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Br?nsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.     

Unilamellar vesicles as potential capreomycin sulfate carriers: Preparation and physicochemical characterization

The aim of this work was to evaluate unilamellar liposomes as new potential capreomycin sulfate (CS) delivery systems for future pulmonary targeting by aerosol administration. Dipalmitoylphosphatidylcholine, hydrogenated phosphatidylcholine, and distearoylphosphatidylcholine were used for liposome preparation. Peptide-membrane interaction was investigated by differential scanning calorimetry (DSC) and attenuated total internal reflection Fourier-transform infrared spectroscopy (ATIR-FTIR). Peptide entrapment, size, and morphology were evaluated by UV spectrophotometry, photocorrelation spectroscopy, and transmission electron microscopy, respectively. Interaction between CS and the outer region of the bilayer was revealed by DSC and ATIR-FTIR. DSPC liposomes showed enhanced interdigitation when the CS molar fraction was increased. Formation of a second phase on the bilayer surface was observed. From kinetic and permeability studies, CS loaded DSPC liposomes resulted more stable if compared to DPPC and HPC over the period of time investigated. The amount of entrapped peptide oscillated between 10% and 13%. Vesicles showed a narrow size distribution, from 138 to 166 nm, and a good morphology. These systems, in particular DSPC liposomes, could represent promising carriers for this peptide.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Intercalation compounds of hydrotalcite-like anionic clays with anti-inflammatory agents,II: Uptake of diclofenac for a controlled release formulation

The purpose of this study was to investigate whether hydrotalcite is able to intercalate diclofenac, a nonsteroidal anti-inflammatory drug, and release it in a controlled manner. Layered Mg−Al hydrotalcite in the chloride form was used as a host, and the intercalation compound was prepared by Cl⁻/diclofenac anionic exchange. Drug release from the intercalation compound was performed in vitro in simulated intestinal fluid at pH 7.5 according to USP 24 and in a pH 7.0 solution designed to mimic the ionic conditions of the small intestine. Results from the intercalation process show that hydrotalcite is able to intercalate diclofenac with a simple procedure and with a good drug loading (55% wt/wt). The in vitro drug release was remarkably lower than that from the corresponding physical mixture at both pH 7.5 and pH 7.0. In the latter case, the release was not complete at 24 hours. The kinetic analysis shows the importance of the diffusion through the particle in controlling the drug release rate. The obtained results show that hydrotalcite may be used to prepare modified release formulations.     

Composition,Antibiofilm, and Antibacterial Potential of Volatile Oils from Geopropolis of Different Stingless Bees’ Species**

We aimed to characterize and investigate the antibacterial potential of the native stingless bees geopropolis volatile oils (VO) for the search of potentially new bioactive compounds. Geopropolis samples from Melipona bicolor schencki, M. compressipes manaosensis, M. fasciculata, M. quadrifasciata, M. marginata and M. seminigra merrillae were collected from hives in South Brazil. VO were obtained by hydrodistillation and characterised by gas chromatography coupled to mass spectrometry (GC/MS). Antimicrobial activity was assessed by microplate dilution method. The lowest MIC against cell walled bacteria was 219±0 μg mL⁻¹ from M. quadrifasciata geopropolis VO with Staphylococcus aureus. The M. b. schencki geopropolis VO minimal inhibition concentration (MIC) was 424±0 μg mL⁻¹ against all the mycoplasma strains evaluated. Fractionation resulted in the reduction of 50 % of the MIC value from the original oil. However, its compounds’ synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25 % eradication activity and 13.20 % inhibition of biofilm formation after 24 h for one subfraction at 2× its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity.     

Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapses of HPV-induced tumors at different anatomical sites

The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8⁺ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8⁺ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.     

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.     

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

Insulin inhibits platelet-derived growth factor-induced cell proliferation

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.     

Placebos and painkillers: is mind as real as matter?

Considerable progress has been made in our understanding of the neurobiological mechanisms of the placebo effect, and most of our knowledge originates from the field of pain and analgesia. Today, the placebo effect represents a promising model that could allow us to shed new light on mind-body interactions. The mental events induced by placebo administration can activate mechanisms that are similar to those activated by drugs, which indicates a similarity between psychosocial and pharmacodynamic effects. These new neurobiological advances are already changing our conception of how clinical trials and medical practice must be viewed and conducted.