Biochemical detection of esterases in the adult female integument of organophosphate-resistant Boophilus microplus (Acari: Ixodidae)

Esterase activity was present in the integument of adult female Boophilus microplus (Canestrini) ticks that are resistant to organophosphates (OP). Three esterases were purified from adult integument, which hydrolyze the substrates p-nitrophenylacetate and beta-naphthyl acetate after comparison of OP-resistant strain and an OP-susceptible strains. The esterases purified by ion-exchange chromatography were characterized using different esterase inhibitors; eserine sulfate, diethyl p-nitrophenyl phosphate (paraoxon), para-hydroxyl-mercuribenzoate (pHMB), and diisopropylphosphofluoridate (DFP). All of the esterases had a molecular mass of 64 Kd (PAGE), but were characterized based on the esterase inhibitor effects as a B-esterase with beta-naphthyl acetate affinity, a carboxylesterase with beta-naphthyl acetate and p-nitrophenyl acetate affinity, and one A-Esterase (nonspecific esterase) with p-nitrophenyl acetate affinity. The described esterases are an important detoxification mechanism in B. microplus ticks at the integument. We describe also a microplate biochemical assay for the detection of esterase activity in the tick integument, potentially a useful tool to detect esterase-mediated OP resistance in B. microplus ticks.     

Optimedin: a novel olfactomedin-related protein that interacts with myocilin

Mutations in the MYOC gene may lead to juvenile open-angle glaucoma with high intraocular pressure, and are detected in about 4% of people with adult onset glaucoma. Most of these mutations are found in the third exon of the gene encoding the olfactomedin-like domain located at the C terminus of the protein. Another olfactomedin-related protein, known as noelin or pancortin, is involved in the generation of neural crest cells. Here we describe the identification of a novel olfactomedin-related gene, named optimedin, located on chromosome 1p21 in humans. Optimedin and noelin are both expressed in brain and retina. However, unlike noelin, rat optimedin is also highly expressed in the epithelial cells of the iris and the ciliary body in close proximity to the sites of Myoc expression. In the human eye, optimedin is expressed in the retina and the trabecular meshwork. Both optimedin and myocilin are localized in Golgi and are secreted proteins. The presence of mutant myocilin interferes with secretion of optimedin in transfected cells. Optimedin and myocilin interact with each other in vitro as judged by the GST pulldown, co-immunoprecipitation and far-western binding assays. The C-terminal olfactomedin domains are essential for interaction between optimedin and myocilin, while the N-terminal domains of both proteins are involved in the formation of protein homodimers. We suggest that optimedin may be a candidate gene for disorders involving the anterior segment of the eye and the retina.     

Soluble Antiapoptotic Molecules and Immune Activation in Chronic Heart Failure and Unstable Angina Pectoris

Programmed myocyte cell death and activation of the immune system have been shown to occur in patients with congestive heart failure. Besides, unstable angina episodes are likely to be associated with immune activation. Our aim was to evaluate the role of changes in circulating levels of soluble Fas (sFas), suggestive of an enhanced inhibitory response to ongoing apoptosis, and soluble IL2 receptor (sIL2-R), indicative of T-lymphocyte activation, in chronic heart failure and unstable angina pectoris. Thirty patients affected by chronic heart failure (20 idiopathic and 10 ischemic cardiomyopathy) and 13 patients with unstable angina were evaluated. Twenty healthy individuals matched for age and gender were used as controls. A complete biochemical determination of indexes of myocardial damage including cardiac troponin I (cTnI) and creatine kinase (MB/CK) was performed. The results demonstrated that mean levels of sFas and sIL2-R were significantly increased in patients affected by chronic heart failure and unstable angina and were not associated with changes in renal function or with serum levels of cTnI. Highest values of sFas were found in NYHA class IV patients (IV NYHA class = 7.39 ± 0.52 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and more elevated in idiopathic than in ischemic cardiomyopathy (3.64 ± 0.40 vs. 1.82 ± 0.37 ng/ml; P < 0.01). Moreover, in chronic heart failure patients sFas and ejection fraction were negatively correlated (P = 0.01), whereas sFas and sIL2-R were positively correlated (P < 0.01). In unstable angina patients too, sFas and sIL2-R appeared to be correlated (P = 0.03); whereas sFas (angina group = 3.18 ± 0.39 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and sIL2-R (angina group = 0.46 ± 0.11 vs. controls = 0.00 UI/ml; P < 0.01) were higher in angina group than in controls. In most of the cases, the increase of sFas was associated with comparable changes in sIL2-R serum levels, indicating that the activation of Fas system is strictly associated with autoimmune–inflammatory reactions. This phenomenon, both in chronic heart failure and in unstable angina, occurs in the absence of biochemical evidences of myocardial damage and seems to parallel the activation of T cell. Soluble Fas could have a role in sustaining inflammatory response and in prolonging the detrimental effects correlated with it in chronic heart failure and angina pectoris.     

A new CARD15 mutation in Blau syndrome

The caspase recruitment domain gene CARD15/NOD2, encoding a cellular receptor involved in an NF-kappaB-mediated pathway of innate immunity, was first identified as a major susceptibility gene for Crohn's disease (CD), and more recently, as responsible for Blau syndrome (BS), a rare autosomal-dominant trait characterized by arthritis, uveitis, skin rash and granulomatous inflammation. While CARD15 variants associated with CD are located within or near the C-terminal leucine-rich repeat domain and cause decreased NF-kappaB activation, BS mutations affect the central nucleotide-binding NACHT domain and result in increased NF-kappaB activation. In an Italian family with BS, we detected a novel mutation E383K, whose pathogenicity is strongly supported by cosegregation with the disease in the family and absence in controls, and by the evolutionary conservation and structural role of the affected glutamate close to the Walker B motif of the nucleotide-binding site in the NACHT domain. Interestingly, substitutions at corresponding positions in another NACHT family member cause similar autoinflammatory phenotypes.     

Multiple pathways of cell invasion are regulated by multiple families of serine proteases

The complex process of tumor invasion requires the coordinated expression and activity of cell-substratum adhesive interactions and of cell-associated protease systems, which destroy the extracellular matrix (ECM), in order to enable the invading cells to simultaneously grip and destroy the anatomical barriers that control cell spreading. A number of data indicate that such a `grip and go' process may be performed by an enlarging series of cell membrane-associated serine proteases and serine protease receptors, which provide the invasive cells with a functional unit (the protease and its receptor), able to mediate cell-substratum adhesion through specific receptor domains, to proteolytically degrade ECM and to deliver into the cell signals that up-regulate the expression either of the protease/receptor complex, or of other adhesion molecules, such as integrins. There is evidence that some proteases and protease receptor expression are under the control of tumor hypoxia, which is the result of an imbalance in oxygen supply and demand. The urokinase-type plasminogen activator (u-PA) receptor (u-PAR) is under hypoxic control and cooperates with other serine proteases of the blood coagulation pathways that may extravasate in the tumor milieu as a result of hypoxia-simulated increase of vessel permeability. Other serine proteases and their receptors cooperate with the cell-associated fibrinolytic system to promote cell invasion. Among these, tissue factor and its ligand coagulation factor VII, thrombin and its protease-activated receptors, and type II trans-membrane serine proteases seem to play a crucial role. This Review takes into consideration the complex scenario of the single serine proteases and related receptors that are involved in cell invasion, as well as the protease receptor/adhesion molecule interplay which is necessary to focus the cell surface-driven proteolysis where adhesion provides a grip to the invading cell. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reversible G(1) arrest by dimethyl sulfoxide as a new method to synchronize Chinese hamster cells

Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, also induces a reversible G(1) arrest in many cell lines. We recently showed that DMSO induces a G(1) phase arrest in Chinese hamster ovary (CHO) cells, by restoring contact inhibition and preventing high density-dependent apoptosis. CHO cells are frequently used in cell biology and mutagenesis studies due to their good growth capacity and ease of manipulation but are very difficult to synchronize by serum starvation since they detach from monolayers when they reach confluence. In this study we investigated the possibility of using DMSO to reversibly synchronize CHO cells in the G(1) phase of the cell cycle and analysed whether toxic effects follow the arrest using growth curve, sister chromatid exchange and micronuclei assays. We carried out a kinetic analysis of the arrest by DMSO and re-entry into the cell cycle after drug release by cytofluorimetric analysis of DNA content and bromodeoxyuridine incorporation. We show that CHO cells are efficiently and reversibly arrested in G(1) by DMSO in concentrations ranging between 1 and 2%. In our experiments, >90% of cells grown for 96 h in presence of the drug were arrested in G(1) and synchronously re-entered S phase approximately 8-12 h after release. Furthermore, expression levels of p27 were down-regulated during G(1) progression and cyclin D3 and E expression patterns were similar to those observed after serum starvation. No detectable cytotoxicity or genetic damage were induced in G(1) released cells as revealed by the tests employed. Our results show that DMSO is a very powerful inducer of G(1) synchronization in CHO cells without detectable cytotoxic or genetic effects in cell populations released from G(1) arrest. DMSO synchronization represents a model system in which to analyse protein activities regulating G(1) progression and investigate the response of G(1) cells to mutagen treatments.     

Identification, characterization and biological activity of oxytocin receptor in the developing human penis

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.     

Viral etiopathogenesis of Sjögren's syndrome: role of the hepatitis C virus

Patients with hepatitis C virus (HCV) chronic infection present some extrahepatic manifestations that may mimic the clinical, immunologic and histological manifestations of primary Sj?gren's syndrome (SS). Thus, HCV patients with sicca symptomatology and positive autoantibodies could be misdiagnosed as a 'primary' SS. Nevertheless, there are several clinical and immunologic features that could help us differentiate both processes.     

Distribution of tetracycline resistance genes in genotypically related and unrelated multiresistant Acinetobacter baumannii strains from different European hospitals

The presence of tetracycline (TET) resistance genes was investigated in 49 genotypically related and unrelated multidrug-resistant Acinetobacter baumannii (MDRAB) strains from European hospitals including representatives of pan-European clones I and II. Except for one strain, all MDRAB strains displayed resistance to tetracycline (MIC range of 16 to > 512 microg/ml) but were susceptible (MIC < 4 microg/ml) or exhibited intermediate resistance (MIC of 4-8 microg/ml) to minocycline (MIN). In 37 strains, either tet(A) or tet(B) was detected and one of these strains possessed both tet(A) and tet(M). In addition, all MDRAB strains contained the aspecific efflux gene adeB irrespectively of whether they harbored tet genes or not. Repetitive DNA element (rep)-PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] revealed that strains previously assigned to pan-European clones I and II were grouped into two separate clusters. In addition, these clusters also contained strains that had not been typed previously, indicating that (GTG)5-PCR is a valuable method for recognizing putative new members of MDRAB clones. Most, but not all, members of clones I and II were linked to the presence of either tet(A) or tet(B) and displayed different levels of TET resistance with MIC values of 32 to > 512 microg/ml and > 512 microg/ml, respectively. Of these two genes only tet(B) encodes an efflux of both TET and MIN, which was reflected by the relatively high MIC values for MIN (4 microg/ml) shown by the majority of the tet(B)-carrying clone II strains as opposed to the low MIC values for MIN (< 1 microg/ml) displayed by most tet(A)-containing clone I strains. Collectively, our phenotypic and genotypic resistance data support the therapeutic evaluation of second-generation tetracyclines like MIN as promising agents for treating MDRAB infections.