Application of saccharose as copper(II) ligand for electroless copper plating solutions

Saccharose, forming sufficiently stable complexes with copper(II) ions in alkaline solutions, was found to be a suitable ligand for copper(II) chelating in alkaline (pH>12) electroless copper deposition solutions. Reduction of copper(II)-saccharose complexes by hydrated formaldehyde was investigated and the copper deposits formed were characterized. The thickness of the compact copper coatings obtained under optimal operating conditions in 1h reaches ca. 2 microm at ambient temperature. The plating solutions were stable and no signs of Cu(II) reduction in the bulk solution were observed. Results were compared with those systems operating with other copper(II) ligands.     

OPA1 cleavage depends on decreased mitochondrial ATP level and bivalent metals

OPA1, an intra-mitochondrial dynamin GTPase, is a key actor of outer and inner mitochondrial membrane dynamic. OPA1 amino-terminal cleavage by PARL and m-AAA proteases was recently proposed to participate to the mitochondrial network dynamic in a DeltaPsi(m)-dependent way, and to apoptosis. Here, by an in vitro approach combining the use of purified mitochondrial fractions and mitochondrial targeting drugs, we intended to identify the central stimulus responsible for OPA1 cleavage. We confirm that apoptosis induction and PTPore opening, as well as DeltaPsi(m) dissipation induce OPA1 cleavage. Nevertheless, our experiments evidenced that decreased mitochondrial ATP levels, either generated by apoptosis induction, DeltaPsi(m) dissipation or inhibition of ATP synthase, is the common and crucial stimulus that controls OPA1 processing. In addition, we report that ectopic iron addition activates OPA1 cleavage, whereas zinc inhibits this process. These results suggest that the ATP-dependent OPA1 processing plays a central role in correlating the energetic metabolism to mitochondrial dynamic and might be involved in the pathophysiology of diseases associated to excess of iron or depletion of zinc and ATP.     

Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.     

Water as a complex system: its role in ligand diffusion, general anesthesia, and sleep

The work and inspiration of Robert Rosen is stated and expressed in personal tones. The concept of passages through water (H2O) near protein surfaces is reviewed in terms of its influence on ligand diffusion to an effector. This is offered as a target for interference by a non-specific general anesthetic agent. In view of the similarities between this anesthetic state and sleep, this mechanism is proposed to be operative for the sleep/wake states. Based on this mechanism and other factors, nitrogen (N2) is proposed as an exogenous sleep factor.     

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeF_x) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeF_x is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, k_obs, for the formation of E2P decreases with [Pi] whereas that of E2BeF_x increases with [BeF_x]. This might wrongly be taken as evidence of a mechanism where the binding of BeF_x induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeF_x follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na⁺. Although E2P and E2BeF_x are able to form states with 2 occluded Rb⁺, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb⁺ is much lower in E2BeF_x than in E2P. The higher rates of Rb⁺ occlusion and deocclusion observed for E2BeF_x, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeF_x mimics the E2P ground state.     

Propionibacterium acnes induces acute TNFα-mediated apoptosis of hepatocytes followed by inflammatory T-cell-mediated granulomatous hepatitis in mice

The CD3+/TCR+ T-cell-mediated hepatic inflammation induced byPropionibacterium acnes could be divided into an acute and a chronic phase. The acute phase occurred within 72 h after injection and displayed hepatic apoptosis. Anti-TNF antibody inhibited both theP. acnes-induced hepatic apoptosis and lymphocyte infiltration seen in this phase, indicating the involvement of this cytokine. Thereafter, a chronic phase was manifested from days 7 to 14 after injection. It was characterized as granulomatous inflammation admixed with apoptosis of infiltrating lymphocytes and some hepatocytes. Immunohistochemical staining showed that the infiltrating lymphocytes displayed TNF, TNF type I receptor and a variety of cytokines including IL-1, IL-4, IL-6, IL-10, IFN or IL-12. Interestingly, in naive mice, the arteries in the liver constitutively expressed IFN. Its expression appeared to be substantially increased at 48 h, decreased at 72 h, and increased again on day 14 afterP. acnes injection. Furthermore, Fas or FasL was only detected on the lymphocytes within the granuloma. We conclude thatP. acnes can induce a TNF-mediated acute hepatic apoptosis which subsequently progress to a T-cell-mediated granulomatous hepatitis with increased expression of multiple cytokines and Fas/FasL.     

Influence of dietary protein on insulin-like growth factor binding proteins in the chicken

We determined the effect of dietary protein on the distribution of insulin-like growth factor (IGF) binding proteins in chicken plasma. Three groups of male broilers (n=6 per group) were fed (ad libitum) isocaloric diets containing 12, 21 or 30% dietary protein. Birds were fed respective diets beginning at 7 days of age and killed at 28 days. No differences were observed between adequate (21%) and high (30%) protein intakes for any of the parameters investigated (growth criteria, plasma levels of IGF-I, growth hormone or IGF-binding proteins). Feeding protein deficient diets (12%) resulted in a 34% decrease in body weight, 17% decrease in feed intake and a 39% increase in feed/gain ratio. IGF-binding proteins in plasma samples were separated by SDS-PAGE and transferred to nitrocellulose sheets. Nitrocellulose blots were probed with [¹²⁵I]chicken IGF-II. Four regions of binding activity corresponding to 70, 43, 30 and 24 kDa were observed in all samples. Birds consuming 12% dietary group protein had less than 50% of the 43-kDa binding activity of birds consuming 21 or 30% dietary protein. The 30-kDa binding activity was 42% lower in the 12% dietary protein group compared to birds consuming adequate protein. In contrast, 70- and 24-kDa binding activities were not influenced by dietary protein. Chickens consuming 12% dietary protein had higher levels of growth hormone and lower levels of IGF-I than those consuming 21 or 30% dietary protein. These data indicate that in chickens, the circulating levels of at least two independent IGF-binding proteins are influenced by dietary protein.     

Fas-mediated apoptosis is attenuated in preneoplastic GST-P-positive hepatocytes isolated from diethylnitrosamine-treated rats

Previous reports have indicated that apoptosis is selectively decreased in enzyme-altered foci (EAF) in the livers of rats treated with a carcinogen. Here we have investigated the effects of an anti-Fas antibody (anti-Fas Ab) on EAF cells in vitro. Hepatocytes were isolated from rats treated repeatedly with diethylnitrosamine (DEN), whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Subsequently, primary cultures of GST-P-positive and GST-P-negative hepatocytes were established and exposed to anti-Fas Ab. Anti-Fas Ab (4 g/ml) preferentially induced apoptosis in GST-P-negative cells. Furthermore, GST-P-positive cells were shown to be resistant to p53-mediated apoptosis. We conclude that EAF hepatocytes are resistant to Fas-mediated apoptosis in vitro. This lack of response may explain the selective decrease in apoptosis in EAF.