Pravastatin up-regulates transforming growth factor-beta1 in THP-1 human macrophages: effect on scavenger receptor class A expression

Statins have been shown to interact with several monocyte/macrophage functions. We tested the effect of pravastatin on transforming growth factor-beta1 (TGF-beta1) production and its possible involvement in scavenger receptors class A (SRA) expression in human THP-1 cells. TGF-beta1s biological activity in THP-1 cell conditioned medium, evaluated by luciferase activity of transfected cell with a TGF-beta responsive promoter, was increased in a dose-dependent manner after incubation with pravastatin (1-20 microM). Pravastatin (1-20 microM) induced a dose-dependent increase in TGF-beta1 mRNA expression and protein production in THP-1 cells. PMA-induced SRA gene and protein expression was suppressed by pravastatin with a mean 3-fold decrease at 10 microM. This last effect was reversed by a mouse monoclonal anti-TGF-beta1 neutralizing antibody. PD98059, a specific inhibitor of MAP kinase cascade, completely reversed pravastatin-induced SRA down-regulation. p44 and p42 isoforms showed a dose-dependent phosphorylation after treatment with pravastatin (1-20 microM) which was inhibited by a mouse monoclonal anti-TGF-beta1 antibody. Our results demonstrate that pravastatin significantly up-regulates TGF-beta1 expression which may be in involved in down-regulation of SRA expression in THP-1 cell cultures. A new pathway for pravastatin effects in atherogenesis can be suggested.     

The herpes simplex virus JMP mutant enters receptor-negative J cells through a novel pathway independent of the known receptors nectin1, HveA, and nectin2

The herpes simplex virus type 1(JMP) [HSV-1(JMP)] mutant was selected for its ability to grow and form plaques in receptor-negative J cells. It enters J cells through a novel gD-dependent pathway, independent of all known HSV receptors, nectin1, nectin2, and HveA. Evidence that the pathway is dependent on a nectin3 binding site on HSV-1(JMP) and requires three mutations in gD rests on the following. We derived monoclonal antibodies to nectin3 and show that J cells express nectin3. HSV-1(JMP) entry and cell-to-cell spread were inhibited by soluble nectin3-Fc, demonstrating that virions carry a binding site for nectin3. The site is either directly involved in HSV-1(JMP) entry, or nectin3 binding to its site affects the gD domains involved in entry (entry site). HSV-1(JMP) entry and cell-to-cell spread in J cells were also inhibited by soluble nectin1-Fc, showing that the nectin1 binding site on gD(JMP) overlaps with the entry site or that nectin1 binding to gD affects the entry site. gD(JMP) carries three mutations, S140N, R340H, and Q344R. The latter two lie in the C tail and are present in the parental HSV-1(MP). HSV-1 strain R5000 carrying the S140N substitution was not infectious in J cells, indicating that this substitution was not sufficient. We constructed two recombinants, one carrying the three substitutions and the other carrying the two C-tail substitutions. Only the first recombinant infected J cells with an efficiency similar to that of HSV-1(JMP), indicating that the three mutations are required for the novel entry pathway. The results highlight plasticity in gD which accounts for changes in receptor usage.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

The concentration dependent biphasic effect of leptin on endogenous cholesterol synthesis in human monocytes

In human monocytes 100 ng/mL leptin increased both statin-inhibitable free radical and cholesterol production in vitro. In our recent study, we aimed to elucidate the concentration dependence of observed leptin-effect. Following leptin stimulation cholesterol synthesis was measured in the presence of inhibitors to determine affected signal pathways. Leptin at low (10-100 ng/mL) concentrations increased [(14)C]acetate incorporation, whereas at 250 ng/mL and higher concentrations it suppressed cholesterol synthesis. HMG CoA reductase, phosphatidyl-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) were involved in mediating leptin effects at low concentrations, whereas the cholesterol synthesis suppression was abolished by inhibitors of protein kinase C (PKC) and PI3K.     

Design, synthesis, and biological evaluation of pirenzepine analogs bearing a 1,2-cyclohexanediamine and perhydroquinoxaline units in exchange for the piperazine ring as antimuscarinics

Pirenzepine (2) is one of the most selective muscarinic M(1) versus M(2) receptor antagonists known. A series of 2 analogs, in which the piperazyl moiety was replaced by a cis- and trans-cyclohexane-1,2-diamine (3-6) or a trans- and cis-perhydroquinoxaline rings (7 and 8) were prepared, with the aim to investigate the role of the piperazine ring of 2 in the interaction with the muscarinic receptors. The structural change leading to compounds 3-6 abolished in binding assays the muscarinic M(1)/M(2) selectivity of 2, due to an increased M(2) affinity. Rather, compounds 3-6 displayed a reversed selectivity showing more affinity at the muscarinic M(2) receptor than at all the other subtypes tested.     

Identification of the Nucleotidase Responsible for the AMP Hydrolysing Hyperactivity Associated with Neurological and Developmental Disorders

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5′-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5′-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5′-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5′-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.     

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.     

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.