Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a–k inspired by 2-BFI scaffold to assess imidazoline molecules as D₂-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D₂-like potency comparable to that of dopamine.     

Immunoregulatory role of lactoferrin-lipopolysaccharide interactions

Lactoferrin (Lf) is a mammalian exclusive protein widely distributed in milk and exocrine secretions exhibiting multifunctional properties. Many of the proven or proposed functions of Lf, apart from its iron binding activity, depend on its capacity to bind to other macromolecules. Lf can bind and sequester lipopolysaccharide (LPS), thus preventing pro-inflammatory pathway activation, sepsis and tissue damage. However, the interplay between Lf and LPS is complex, and may result in different outcomes, including both suppression of the inflammatory response and immune activation. These findings are critically relevant in the development of Lf-based therapeutic interventions in humans. Understanding the molecular basis and functional consequences of Lf-LPS interaction will provide insights for determining its role in health and disease.     

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.     

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.     

Evolution of ragweed pollen concentrations, sensitization and related allergic clinical symptoms in Parma (northern Italy)

The pollen released in the atmosphere by the ragweed represents a question of public health in several European countries. In Italy, the ragweed is mostly distributed in the North. In our region (Emilia Romagna), the presence of ragweed was not described yet if not occasionally, but this plant is thriving well in the North of the Po river. The aim of our study was to estimate the concentration trends of ragweed pollens in the air of Parma starting from 1992 until 2008 and to describe the clinical related situation. The aerobiological surveillance was made with the methods standardized by the Italian Association of Aerobiology. We analyzed 19,468 outpatients affected by respiratory disease. The patients studied address our clinical Center, mainly with pathologies respiratory, most of them with allergic origin. To detect the existence of significant trends and correlations since, we used the non-parametric tests with SPSS software. Our observations showed that since 1995, the year until when pollens of ragweed were only sporadically observed in the air of Parma, there has been a significant increase in ragweed pollens. Among the patients addressed at our clinical Center, 876 patients had positive SPT (skin prick test) for ragweed pollen with respiratory illnesses, all polysensitized. Besides, we found a significant increase in patients with positive SPT for the ragweed, and among these, the increase in the asthma has been significant. On the basis of our results, we expect, in the absence of intervention from public authorities, a more significant increase in the positive subjects and an aggravation of the symptoms related to the presence of ragweed pollen in the air of Parma.     

LF immunomodulatory strategies: mastering bacterial endotoxin

Lactoferrin (LF), an iron-binding glycoprotein expressed in most biological fluids, represents a major component of mammalian innate immune system. The multiple activities of LF rely not only on its capacity to bind iron but also to interact with molecular and cellular components of both the host and pathogens. LF can bind and sequester lipopolysaccharide thus preventing proinflammatory pathway activation, sepsis, and tissue damage. However, the interplay between LF and lipopolysaccharide is complex and may lead to different outcomes including both the suppression of inflammatory response and immune activation. Understanding the molecular basis and the functional consequences of this complex interaction is critically relevant in the development of LF-based therapeutic interventions in humans.     

Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8(+) T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20(+) B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8(+) T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.     

Synthesis, characterization, crystal structures and in vitro antistaphylococcal activity of organotin(IV) derivatives with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine

New organotin(IV) complexes of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) with 1:1 and/or 1:2 stoichiometry were synthesized and investigated by X-ray diffraction, FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H and ¹³C NMR spectroscopy, in solution. Moreover, the crystal and molecular structures of Et₂SnCl₂(dbtp)₂ and Ph₂SnCl₂(EtOH)₂(dptp)₂ are reported. The complexes contain hexacoordinated tin atoms: in Et₂SnCl₂(dbtp)₂ two 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine molecules coordinate classically the tin atom through N(3) atom and the coordination around the tin atom shows a skew trapezoidal structure with axial ethyl groups. In Ph₂SnCl₂(EtOH)₂(dptp)₂ two ethanol molecules coordinate tin through the oxygen atom and the 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the OH group of the ethanol moieties; Ph₂SnCl₂(EtOH)₂(dptp)₂ has an all-trans structure and the C-Sn-C fragment is linear. On the basis of Mössbauer data, the 1:2 diorganotin(IV) complexes are advanced to have the same structure of Et₂SnCl₂(dbtp)₂, while Me₂SnCl₂(dptp)₂ to have a regular all-trans octahedral structure. A distorted cis-R₂ trigonal bipyramidal structure is assigned to 1:1 diorganotin(IV) complexes. The in vitro antibacterial activities of the synthesized complexes have been tested against a group of reference pathogen micro-organisms and some of them resulted active with MIC values of 5 μg/mL, most of all against staphylococcal strains, which shows their inhibitory effect.     

Gallstone disease is associated with more severe liver damage in patients with non-alcoholic Fatty liver disease

Background

Nonalcoholic fatty liver disease (NAFLD) and gallstone disease (GD) are both highly prevalent in the general population and associated with obesity and insulin resistance. We aimed to evaluate the prevalence of GD in a cross sectional study of NAFLD patients and to define whether the presence of GD is associated with diabetes and predicts more severe liver disease.

Methodology/Principal Findings

We merged databases of four Liver Units, comprising 524 consecutive biopsy-proven NAFLD (373 males) observed between January 2003 and June 2010. GD was diagnosed in 108 (20%), and 313 cases (60%) were classified by liver biopsy as nonalcoholic steatohepatitis (NASH). The GD subgroup was characterized by a significantly higher prevalence of females, prediabetes/diabetes, abdominal obesity and metabolic syndrome, older age, higher BMI, fasting glucose, HOMA-IR and lower ALT. The prevalence of GD progressively increased with advancing fibrosis and with the severity of necroinflammatory activity (p for trend  = 0.0001 and  = 0.01, respectively), without differences in the severity of steatosis. At multivariate analysis GD was associated with female gender (OR 1.37, 95% CI 1.04–1.8), age (OR 1.027, 95% CI1.003–1.05), fasting glucose (OR 1.21, 95% CI 1.10–1.33) and NASH (OR 1.40,95% CI 1.06–1.89), whereas ALT levels were associated with a lower GD risk (OR 0.98, 95% CI 0.97–0.99). When subjects with cirrhosis were excluded from analysis, the association between GD and fasting glucose, female gender, and NASH was maintained.

Conclusion

Patients with NAFLD have a high prevalence of GD, which characterizes subjects with altered glucose regulation and more advanced liver disease.