Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Paget's disease of bone (PDB). We originally described a methionine-->valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patient's pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype-phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype-phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.     

Biodegradation of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis> diethylaniline in a contaminated aquifer: laboratory- and field-scale evidences

The effectiveness of biosparging to mitigate N,N diethylaniline in aquifer was evaluated by measuring the time course of decrease in concentration of the aforementioned compound in aerobic microcosm experiments. The first-order kinetic constant for N,N diethylaniline aerobic biodegradation was estimated from microcosm data (0.037 ± 0.004 d⁻¹), and the value was consistent with the best-fitting value in the transport and reaction model of the aquifer (0.020 d⁻¹). Furthermore, the biodegradability of the compound was evaluated under anaerobic condition in microcosm experiments, which was supported by field modelling. There was no significant degradation in the anaerobic microcosm experiments, confirming the recalcitrance of N,N diethyl aniline under the aforementioned aquifer condition.     

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.     

Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.     

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.     

AQP1 expression analysis in human diseases: implications for proteomic characterization

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.     

Chemokines and Glial Cells: A Complex Network in the Central Nervous System

Chemokines are small secreted proteins that are essential for the recruitment and activation of specific leukocyte subsets at sites of inflammation and for the development and homeostasis of lymphoid and nonlymphoid tissues. During the past decade, chemokines and their receptors have also emerged as key signaling molecules in neuroinflammatory processes and in the development and functioning of the central nervous system. Neurons and glial cells, including astrocytes, oligodendrocytes, and microglia, have been identified as cellular sources and/or targets of chemokines produced in the central nervous system in physiological and pathological conditions. In this article, we provide an update of chemokines and chemokine receptors expressed by glial cells focusing on their biological functions and implications in neurological diseases.     

Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis

Generalized pustular psoriasis (GPP) is a rare and yet potentially lethal clinical variant of psoriasis, characterized by the formation of sterile cutaneous pustules, neutrophilia, fever and features of systemic inflammation. We sequenced the exomes of five unrelated individuals diagnosed with GPP. Nonsynonymous, splice-site, insertion, and deletion variants with an estimated population frequency of <0.01 were considered as candidate pathogenic mutations. A homozygous c.338C>T (p.Ser113Leu) missense substitution of IL36RN was identified in two individuals, with a third subject found to be a compound heterozygote for c.338C>T (p.Ser113Leu) and a c.142C>T (p.Arg48Trp) missense substitution. IL36RN (previously known as IL1F5) encodes an IL-1 family receptor antagonist, which opposes the activity of the IL-36A and IL-36G innate cytokines. Homology searches revealed that GPP mutations alter evolutionarily conserved residues. Homozygosity for the c.338C>T (p.Ser113Leu) variant is associated with an elevated proinflammatory response following ex vivo stimulation with IL36A. These findings suggest loss of function of IL36RN as the genetic basis of GPP and implicate innate immune dysregulation in this severe episodic inflammatory disease, thereby highlighting IL-1 signaling as a potential target for therapeutic intervention.