Tick species from Africa by migratory birds: a 3-year study in Italy.

The role of resident or migratory birds in dispersal of tick species and tick-borne pathogens is still poorly known in Italy. We report here the results of a 3-year project based on sampling ticks from migratory birds, as well as from the vegetation at three stop-over sites for migrants, namely the islands of Ventotene (Latium), Asinara (Sardinia) and Ustica (Sicily). During the spring seasons from 2017-2019, in total 2681 ticks were collected, 2344 of which were sampled from migratory birds and 337 from the vegetation. Ticks were identified by morphology or by molecular tools when necessary. In total, 16 tick species were identified among which the following were exclusively found on birds: Hyalomma rufipes (43.3%), Hy. truncatum (0.1%), Ixodes frontalis (11.8%), Ix. inopinatus (0.2%), Ix. ricinus (3%), Haemaphysalis punctata (0.08%), Hae. erinacei (0.1%), Amblyomma variegatum (0.08%) and Argas vulgaris 0.1%), whereas five species were exclusively collected from the vegetation: Rhipicephalus bursa (10.5%), Rh. turanicus (5.9%), Rh. sanguineus sensu lato (2%), Rh. pusillus (2.4%), Hae. sulcata (0.08%). Hy. marginatum (10.3%) and Ix. ventalloi (9.3%) were found both on birds and on the vegetation on the island Ustica. It is worth noting that the search for ticks on the vegetation did not detect allochthonous tick species. Although we found several interesting local species and allochthonous ticks like Hy. rufipes, Am. variegatum and Ar. vulgaris on birds, further investigations are needed to better define the possible role of migratory birds in the introduction of ticks and tick-borne diseases in Italy, above all after the evidence of imported ticks positive to Crimean Congo hemorrhagic fever (CCHF) virus in several European countries.

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts.

In addition to its role in invasion and metastasis of several tumors, the multifunctional urokinase receptor uPAR (urokinase plasminogen activator receptor) is directly involved in the growth of several cancer cells in vitro and in vivo. We have compared growth rate and oncogenic transformation in wild-type (wt) or uPAR-/- mouse embryonic fibroblasts (MEFs). Surprisingly, uPAR-/- MEFs grew faster than wt MEFs. This agreed with elevated levels of cell cycle mediators like extracellular signal-regulated protein kinase, p38, AP1 and Cyclin D1. Infection with a uPAR retrovirus reverted the effect, decreasing the growth rate. When MEFs were transformed with H-Ras(V12) and E1A oncogenes, the efficiency of transformation in uPAR-/- MEFs was higher than in wt. UPAR-/- MEFs grew faster at low serum, produced more colonies in agar and produced tumors in vivo in nude mice with a lower latency period. The properties of the heterozygous uPAR+/- MEFs were always intermediate. We conclude therefore that in MEFs uPAR concentration controls cell proliferation and the transforming activity of some oncogenes.

Rheumatoid synovial fibroblasts constitutively express the fibrinolytic pattern of invasive tumor-like cells.

OBJECTIVE: In rheumatoid arthritis (RA) the synovial membrane proliferates and invades the underlying tissues. The cell-associated fibrinolytic system (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor-type 1, PAI-1) is pivotal in cell invasion and proliferation. For this reason, the expression and the role of such enzymatic system was investigated in synovial fibroblasts (SF) of normal and RA patients. METHODS: In SF obtained from RA patients and control subjects, uPA, uPAR and PAI-1 were measured by ELISA of cell lysates and culture medium and by RT-PCR of mRNAs. uPA was also studied by zymography. Proliferation was measured by cell counting and cell invasion with the Boyden chamber. RESULTS: RA-SF over-express uPAR and PAI-1 and are more prone than the normal counterpart to spontaneous and uPA-challenged invasion and proliferation, which are counteracted by antagonists of the fibrinolytic system. CONCLUSIONS: RA-SF display the fibrinolytic pattern and behaviour of invasive tumor-like cells. Antagonists of the fibrinolytic system are able to revert growth and invasion of both normal and RA-SF.

Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Inhibition of contact hypersensitivity reaction to picryl chloride: effect of small molecular weight peptidomimetic compounds possessing inhibitory activity against metalloproteinases.

We have studied the effect of small molecular weight inhibitors of snake venom metalloproteinases (SVMP) and matrix metalloproteinases (MMPs) on the induction and effector phase of the contact hypersensitivity reaction (CHR) in a mouse model. Identification of nonsteroid small molecules is very important for the development of new anti-inflammatory drugs. The compounds that we tested were synthetically modified tripeptides (peptidomimetic compounds) POL-257, POL-509, POL-443, POL-491, and POL-647, with structures based on natural occurring peptides in snake venom. A well-known hydroxamate-based inhibitor of the MMPs, Batimastat (BB-94), was also used. We have shown that these peptidomimetics possess in vitro inhibitory activity against the MMP-2 (gelatinase-A), MMP-9 (gelatinase-B), and MMP-3 (stromelysin). They also inhibit metalloproteinases purified from the venom of Crotalus adamanteus and C. atrox snakes, which are very similar to the so-called A Desintegrine, A Metalloproteinase (ADAMs) enzymes. When injected intraperitonealy before the topical application of the contact sensitizer (picryl chloride) or before the challenge, these compounds significantly inhibited the development of CHR. BB-94 at doses 0.4 and 4 mg/kg before the sensitization or before the challenge almost completely abrogated the reaction. POL-257 and POL-443 were among the most active peptidomimetics tested. They inhibited the inflammatory reaction up to 70-80%, when applied either immediately before sensitization or before challenge. POL-509, a methylated derivative of POL-257, inhibited the CHR to 40-50% when administered at either challenge or sensitization. However, when applied 24 h before the challenge, it completely abrogated the inflammatory reaction. The results show that these small molecular weight peptidomimetic compounds, as well as BB-94, are able to significantly inhibit the CHR. This finding opens possibilities for using metalloproteinase inhibitors in the treatment of allergic contact dermatitis and other inflammatory diseases.

Conformationally constrained analogues of endogenous tripeptide inhibitors of zinc metalloproteinases.

Two diastereomeric furan-2-carbonylamino-3-oxohexahydroindolizino[8,7-b]indole carboxylates, highly constrained analogues of endogenous pyroglutamyl tripeptide inhibitors of snake venom endopeptidases, have been prepared as potential inhibitors of adamalysin II and matrix metalloproteinases. They proved to be inactive against adamalysin II and weak inhibitors of gelatinase A, gelatinase B, stromelysin 1 and human neutrophil collagenase. Evaluation of the mode of binding of the (2R,5S,11bR) isomer in the active site of adamalysin II suggests that the decrease of potency may be due to the reorientation of the acylamino chain in three of the heterocyclic nucleus, to a short contact at the entrance of the S'(1) hydrophobic cleft and to the loss of flexibility of the tetracyclic nucleus in the P'(1), P'(2) region of the inhibitor, which prevents optimal arrangement in the S'(1) specificity subsite.

Transforming growth factor beta-1 stimulates invasivity of hepatic stellate cells by engagement of the cell-associated fibrinolytic system.

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-beta1 (TGF-beta1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-beta1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-beta1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-beta1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-beta1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-beta1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-beta1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-beta1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-beta1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B.

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the neutrophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.

Two crystal structures of human neutrophil collagenase, one complexed with a primed- and the other with an unprimed-side inhibitor: implications for drug design.

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.

Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates.

The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.

Inhibition of adamalysin II and MMPs by phosphonate analogues of snake venom peptides.

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.

Inhibitors of Zn-dependent metallopeptidases: synthesis and activity of N-(2-furoyl)-(Z)-alpha, beta-didehydroleucyl-L-tryptophan.

In order to gain information on structure activity relationships of peptidic inhibitors of Zn-dependent metalloproteinases "hemorrhagins", the conformationally restricted model N-(2-furoyl)-(Z)-alpha,beta-didehydroleucyl-L-tryptophan 2 was synthesized and its activity compared to that of related previously studied substrates. The new model 2 exhibits an inhibitory activity on proteinase II from Crotalus Adamanteus snake venom, sensibly lower than that of related substrates. This result indicates that the reduction of the conformational space due to the presence of the alpha,beta-didehydro-amino acid residue in 2 does not favour the fitting and binding at the enzyme active site.

Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.

The multidomain structure of ceruloplasmin from calorimetric and limited proteolysis studies.

Differential scanning calorimetry has been used to investigate the thermal stability of three different ceruloplasmins (from sheep, chicken, and turtle) in their native state and after limited proteolysis. The three undegraded proteins showed a similar structural organization in three calorimetric domains, although their temperature of unfolding varied from 57.8 degrees C (turtle) to 71.2 degrees C (sheep) to 82.1 degrees C (chicken). The spectroscopic and the catalytic properties were totally lost at temperatures corresponding to the unfolding of the less thermostable domain in the case of sheep and chicken ceruloplasmins and to the unfolding of the most thermostable domain in the turtle protein. Trypsin, but not plasmin, digestion caused a significant decrease of the thermal stability of sheep and chicken ceruloplasmins. Turtle ceruloplasmin was insensitive to both proteases. Comparing the thermodynamic parameters of the sheep protein in its undegraded and cleaved states revealed a mismatch between the three calorimetric domains and the 3-fold internal replication of the primary structure, which is evident in the highly homologous, fully sequenced human protein. Copper removal caused the rearrangement of the molecule in only two calorimetric domains, suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the less thermostable cooperative unit of the native protein.

The role of copper in the stability of ascorbate oxidase towards denaturing agents.

The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.