Divalent Metal Chelates Research Articles

Trivalent immunization with metal-binding proteins confers protection against enterococci in a mouse infection model.

Enterococcus faecalis is ranked among the top five bacterial pathogens responsible for catheter-associated urinary tract infections, wound infections, secondary root canal infections, and infective endocarditis. Previously, we showed that inactivation of either the manganese- and iron-binding (EfaA) or zinc-binding (AdcA and AdcAII) lipoproteins significantly reduced E. faecalis virulence. Here, we explored whether immunization using a multi-valent approach induces protective immunity against systemic enterococcal infections. We found that multi-antigen antisera raised against EfaA, AdcA, and AdcAII displayed similar capacities to initiate neutrophil-mediated opsonization, like their single-antigen counterparts. Further, these antigen-specific antibodies worked synergistically with calprotectin, a divalent host metal chelator, to inhibit the growth of E. faecalis in laboratory media as well as in human sera. Using the Galleria mellonella invertebrate model and mouse peritonitis model, we showed that passive immunization with multi-antigen antisera conferred robust protection against E. faecalis infection, while the protective effects of single antigen antisera were negligible in G. mellonella, and negligible-to-moderate in the mouse model. Lastly, active immunization with the 3-antigen (trivalent) cocktail significantly protected mice against either lethal or non-lethal E. faecalis infections, with this protection appearing to be far-reaching based on immunization results obtained with contemporary strains of E. faecalis and closely related Enterococcus faecium.

Insight into the synthesis, structure affirmation and catalytic efficiency of divalent and trivalent metal chelates of mandelic acid hydrazone derivative.

The current work reports the synthesis of Cr(iii), Mn(ii), Co(ii), Ni(ii) and Cu(ii) chelates of the Schiff base ligand named hydroxy-phenyl-acetic acid (2-hydroxy-naphthalen-1-ylmethylene)-hydrazide with multi-chelation centre toward metal ions. The spectral tools, 1H-NMR, FTIR, mass, UV-vis spectra, and the analytical elemental and thermal analysis, in addition to magnetic moment and conductivity measurements all combined have been applied to conclude the structure and geometry of the synthesized metal complexes. The formed metal chelates have been assured to be formed with the molar compositions of 1 L : 1 M for PANH-Cr, PANH-Mn, PANH-Co, PANH-Ni and 2 L : 1 M for PANH-Cu. All the complexes have been confirmed to be non-electrolytic except the PANH-Mn and PANH-Ni which are 1 : 1 electrolytes. FTIR spectral analysis assured the ligand to act as mono basic bi or tridentate ligand leading to the formation of octahedral complexes with all metals except Cu(ii) complex which assured to has square planar structure. Except PANH-Cr, all the synthesized metal chelates exhibited phenoxazinone synthase like efficacy with varying activity with dramatically high activity for PANH-Mn complex with TOF number of 169.89 h-1.

Crystal structures and physicochemical studies of some novel divalent and trivalent transition metal chelates of N-morpholine-N'-benzoylthiourea

A series of Fe(III), Co(III), Ni(II), Cu(II), Zn(II) and In(III) N-morpholine-N'-benzoylthiourea complexes have been synthesised and characterised by elemental analysis, thermal analysis, infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy and single crystal X-ray crystallography. Thermogravimetric analysis shows that all the complexes undergo a two-step decomposition process except for the iron(III) complex and the indium(III) complexes, which show three-step and one-step decompositions, respectively. The complexes are thermally stable up to approximately 300 °C. The ligand coordinates the various metal ions in a bidentate (L-kO,S) chelating mode, facilitated by deprotonation of the acidic amide (–C(O) N'HC (S)) moiety. This mode of coordination allows for the facile formation of neutral bis/tris-6-membered chelates of type [M(L-kS,O)x] where x = 2 or 3 for divalent or trivalent metal ions, respectively.

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action.

Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.

The effect of divalent metal chelators and cadmium on serum phosphotriesterase, lactonase and arylesterase activities of paraoxonase 1

Paraoxonase 1 (PON1) is calcium dependent enzyme involved in many functions in human body. PON1 is synthesized in the liver and secreted to the bloodstream where bounds high-density lipoproteins (HDL). Association of PON1 with HDL increases the enzyme stability and biological activities. PON1 have three different activities: phosphotriesterase, lactonase and arylesterase. Until now there is now commercial available kits to determine these three PON1 activities. Also there is no date about stability of PON1 in serum after storage condition. We have elaborated the optimal conditions for determination of PON1 activities in serum using manual procedure as well as the best storage temperature of human serum for determination of PON1 activities. We have also confirmed that PON1 in serum is associated with HDL. Additionally we have investigated the effect of D-penicillamine, ethylenediaminetetraacetic acid and cadmium chloride on PON1 activities in human serum. D-penicillamine and ethylenediaminetetraacetic acid in therapeutic doses as well as cadmium chloride in toxic doses decrease PON1 activities in human serum when compared to non-treated serum. D-penicillamine as metal chelator inhibits much stronger PON1 activities than ethylenediaminetetraacetic acid.

Fusaric Acid immunotoxicity and MAPK activation in normal peripheral blood mononuclear cells and Thp-1 cells

Fusaric acid (FA), a food-borne mycotoxin, is a potent divalent metal chelator. The human immune system is complex and susceptible to environmental insult however, the immunotoxity of FA remains unknown. We investigated the immunotoxicity of FA on human peripheral blood mononuclear cells (PBMCs) and Thp-1 cells. FA was cytotoxic to PBMCs (IC50-240.8 μg/ml) and Thp-1 (IC50-107.7 μg/ml) cells at 24 h. FA induced early apoptosis but significantly decreased caspase activity in PBMCs, a characteristic of paraptosis. In Thp-1 cells, FA induced apoptosis and increased caspase −9 and −3/7 activities. In PBMCs, FA maintained mitochondrial membrane potential and decreased protein expression of Bax whilst increasing expression of p-Bcl-2; FA induced oxidative stress and depleted ATP levels in both cell types. In Thp-1 cells, FA increased mitochondrial membrane depolarization and decreased p-Bcl-2 expression. In PBMCs, FA significantly up-regulated the MAPK protein expression of p-ERK and p-JNK but down-regulated p-p38 expression. In Thp-1 cells, FA up-regulated MAPK protein expression of p-ERK whilst p-JNK and p-p38 expression were down-regulated. In conclusion FA induced programmed cell death and altered MAPK signaling in healthy PBMCs and Thp-1 cells strongly suggesting a possible mechanism of FA induced immunotoxicity in vitro.

In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors.

Synthesis, Spectral Studies of Binuclear Divalent Metal Chelates Derived from Benzofuron-2-carbo-hydrazide and 1-Chloro-4-isothiocyanatobenzene

Synthesis, Spectral Studies of Binuclear Divalent Metal Chelates Derived from Benzofuron-2-carbo-hydrazide and 1-Chloro-4-isothiocyanatobenzene

Impact of iron chelators on short-term dissolution of basaltic glass

Although microorganisms seem to play an important role in the alteration processes of basaltic glasses in solution, the elementary mechanisms involved remain unclear in particular with regard to the role of organic ligands excreted by the cells. Two glasses, one with Fe and one without Fe were synthesized to model basaltic glass compositions. Fe in the glass was mostly Fe(III) for enhancing interaction with siderophores, yet with small but significant amounts of Fe(II) (between 10% and 30% of iron). The prepared samples were submitted to abiotic alteration experiments in buffered (pH 6.4) diluted solutions of metal-specific ligands, namely oxalic acid (OA, 10mM), desferrioxamine (DFA, 1mM) or 2,2′-bipyridyl (BPI, 1mM). Element release from the glass into the solution after short term alteration (maximum 1week) was measured by ICP-OES, and normalized mass losses and relative release ratios (with respect to Si) were evaluated for each element in each experimental condition. The presence of organic ligands had a significant effect on the dissolution of both glasses. Trivalent metals chelators (OA, DFA) impacted on the release of Fe3+ and Al3+, and thus on the global dissolution of both glasses, enhancing all release rates and dissolution stoichiometry (release rates were increased up to 7 times for Al or Fe). As expected, the mostly divalent metal chelator BPI interacted preferentially with Ca2+, Mg2+ and Fe2+. This study thus allows to highlight the central roles of iron and aluminium in interaction with some organic ligands in the alteration processes of basaltic glasses. It thus provides a step toward understanding the biological contribution of this fundamental geological process.

English

Seed proteins have been evaluated for their nutritive value and biological activity. Proteases and proteinase inhibitors have been of immense benefit to both agriculture and therapeutic purposes. The seed proteins of Blighia sapida was evaluated for both structure and biological activity in this study. The matured dried seeds were pulverized and sequentially extracted using 10 mM Tris/HCl pH 7.4, 10 mM ammonium acetate and 10 mM sulphuric acid. Crude protein extracts were concentrated and the protein concentrations were estimated. Proteins were purified by 70% ammonium sulphate precipitation, Sephadex G50 reversed phase chromatography and finally by HPLC on a C18 column. Two bands were obtained from SDS-PAGE electrophoresis and they were identified by ESI/MS using in gel tryptic digestion. The seed protein from B. Sapida consists of two single polypeptide chains each with mass of about 24 to 27 KDa as established by a combination of SDS-PAGE and ESI/MS. Proteins exhibited protease activity, which was confirmed by zymography. Protease activity was characterized for effect of temperature, pH, divalent metal ions and chelating agents. The crude protein from the seed of B. sapida showed antimicrobial activity and the antifungal activity was comparable with the reference drug, Ticonazole. Keywords: Blighia sapida, chromatography, protease activity, in-gel trypsin digestion/mass spectrophotometry, antimicrobial activity. African Journal of Biotechnology , Vol 13(29) 2996-3007

Synthesis, structural characterization, electrochemical and biological studies on divalent metal chelates of a new ligand derived from pharmaceutical preservative, dehydroacetic acid, with 1,4-diaminobenzene

Cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) complexes of new 3-acetyl-4-[(4-aminophenyl)amino]-6-methyl-2H-pyran-2-one (HL1) derived from dehydroacetic acid and 1,4-diaminobenzene were prepared and characterized. The structural features were determined from their elemental analyses, 1H, and 13C-NMR spectra, molar conductivities, magnetic moments, IR, UVvis. spectra, thermal analyses (D.T.A. and T.G.A.) and E.S.R. measurements. Their magnetic susceptibility measurements and low conductance data provide evidence for the mono- or dimeric and non-electrolytic nature of the solid complexes. The E.S.R. spectra of copper(II) complexes show axial type symmetry with covalent or ionic bond character. The electrochemical behavior of the complexes in DMF (dimethylformamide) solvent at 298K was studied. The biological activity of the ligand and its metal(II) complexes was also studied. The obtained complexes showed higher activities than the free ligand in protecting the Egyptian cotton fields from Spodoptera littoralis larvae.

Characterization of Plp, a phosphatidylcholine-specific phospholipase and hemolysin of Vibrio anguillarum

BackgroundVibrio anguillarum is the causative agent of vibriosis in fish. Several extracellular proteins secreted by V. anguillarum have been shown to contribute to virulence. While two hemolysin gene clusters, vah1-plp and rtxACHBDE, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. Here we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis.ResultsThe plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), which has homology to lipolytic enzymes containing the catalytic site amino acid signature SGNH. Hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that Plp has different effects against erythrocytes from different species. His6-tagged recombinant Plp protein (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5–9 and at temperatures of 30-64°C. Divalent cations and metal chelators did not affect activity of rPlp. We also demonstrated that Plp was secreted using thin layer chromatography and immunoblot analysis. Additionally, rPlp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rPlp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. Further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxA and/or vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.ConclusionThe plp gene of V. anguillarum encoding a phospholipase with A2 activity is specific for phosphatidylcholine and, therefore, able to lyse fish erythrocytes, but not sheep erythrocytes. Mutation of plp does not affect the virulence of V. anguillarum in rainbow trout.

Nicotianamine synthase gene family as central components in heavy metal and phytohormone response in maize

Nicotianamine (NA) is an important divalent metal chelator and the main precursor of phytosiderophores. NA is synthesized from S-adenosylmethionine in a process catalyzed by nicotianamine synthase (NAS). In this study, a set of structural and phylogenetic analyses have been applied to identify the maize NAS genes based on the maize genome sequence release. Ten maize NAS genes have been mapped; seven of them have not been reported to date. Phylogenetic analysis and expression pattern from microarray data led to their classification into two different orthologous groups. C-terminal fusion of ZmNAS3 with GFP was found in the cytoplasm of Arabidopsis leaf protoplast. Expression analysis by reverse transcription polymerase chain reaction revealed ZmNAS genes are responsive to heavy metal ions (Ni, Fe, Cu, Mn, Zn, and Cd), and all 10 ZmNAS genes were only observed in the root tissue except of ZmNAS6. The promoter of ZmNAS genes was analyzed for the presence of different cis-element response to all kinds of phytohormones and environment stresses. We found that the ZmNAS gene expression of maize seedlings was regulated by jasmonic acid, abscisic acid, and salicylic acid. Microarray data demonstrated that the ZmNAS genes show differential, organ-specific expression patterns in the maize developmental steps. The integrated comparative analysis can improve our current view of ZmNAS genes and facilitate the functional characterization of individual members.

Inhibition of HIV-1 tat-induced transactivation and apoptosis by the divalent metal chelators, fusaric acid and picolinic acid—Implications for HIV-1 dementia

The HIV-1 transactivator protein tat is pivotal to the pathogenesis of AIDS, exerting its effects on both viral and cellular gene expression. The basic structure of tat protein allows it to be secreted by HIV-1 infected cells and penetrate uninfected cells where it elicits its multifunctional biochemical effects. The main function of tat protein is viral transactivation which leads to the upregulation of transcription through complex interactions with RNA and host cell factors. Since HIV-1 has been widely implicated as a causative agent of HIV-1 dementia, the aim of our study was to investigate the ability of two novel metal chelators, fusaric acid (FA) and picolinic acid (PA) to firstly inhibit HIV-1 tat induced transcription and secondly, to minimize its cytotoxic effects as mediated via apoptosis. Biologically active tat protein is not freely available commercially. We therefore had to produce, isolate and purify our own protein. A cell culture system and flow cytometric techniques were used in our study. Exposure of CEM-GFP cells to exogenous recombinant tat protein induced transcription and apoptosis, and both processes were inhibited by FA and PA at concentrations that alone did not induce any cytotoxicity. Our data suggest that FA and PA may have therapeutic potential in the management of HIV-1 dementia.

Chemicals That Stimulate Iron Uptake – A Novel Approach to Treatment of Iron Deficiency,

Abstract 3180 Iron (Fe) is an essential nutrient required for all cells, especially for erythrocyte hemoglobin synthesis which requires absorption of 1–2 mg of iron from the gastrointestinal tract. Iron deficiency as a result of inadequate dietary uptake has multiple consequences including anemia and abnormal neurologic development in children and is a global public health concern. Enterocytes in the duodenum, the site of iron absorption, can extract about 10% of dietary Fe. Nonetheless for numerous reasons simple iron supplementation has not solved the worldwide epidemic of iron deficiency. We hypothesized that small molecules which could potentiate iron uptake into cells would allow enterocytes to absorb an increased amount of dietary iron and could be beneficial in limiting iron deficiency. To identify molecules that would accelerate Fe uptake we used a high through-put screening system in conjunction with a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. Small molecules that stimulated Fe uptake were defined as causing increased calcein fluorescence quenching compared to Fe alone. K562 cells were exposed to 0.1 μM calcein for 10 minutes, thoroughly washed, and 1 × 105 cells plated into each well of multiple 96-well plates. After equilibration of the plates at 37° C, aliquots of the individual components of an in-house chemical library of ∼12,000 compounds dissolved in DMSO were screened in duplicate or triplicate and fluorescence measurements made at 0 and 30 min after addition of 10 μM FeNH4SO4 in a Synergy IV plate reader. 30 chemicals were identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose response curves and by assaying the effect on non-transferrin bound 55Fe uptake. None of the stimulators were cytotoxic for up to at least 3 days. The lead compound, LS081, had an IC50 = 1.22 ± 0.48 μM for 55Fe uptake in K562 cells compared to controls. LS081 was also used to examine the iron uptake in Caco2 cells grown in bicameral chambers, a model system to study intestinal iron absorption. LS081 significantly increased 55Fe uptake into Caco2 cells with a very rapid influx of 55Fe in the first 5 min after Fe was offered to the apical surface followed by a ∼ 4-fold increased uptake over the next 90 min. 55Fe transport across the basolateral surface into the basal chamber also increased ∼ 4 fold. The increased 55Fe transport in caco2 cells is more prominent at lower pH of 5.5 compare to pH 7.5 suggesting LS081 acted on a common divalent metal uptake pathway. Mice treated with LS081 + ferric ammonium citrate via oral gavage for two weeks significantly increased (p < 0.001 by unpaired t-test compared to ferric ammonium citrate alone) the level of ferritin, the iron storage protein, in the liver, demonstrating the absorption of LS081 from intestinal cells. In summary, using high through-put screening technique we identified small molecules that stimulate iron uptake and could be used as a drug for iron deficiency. Disclosures: No relevant conflicts of interest to declare.

Abstract 3530: Chemicals that stimulate iron uptake: a novel approach to cancer treatment

Abstract Cancer cells have increased levels of transferrin receptor and lower levels of ferritin, an iron deficient phenotype that has led to the use of iron chelators to further deplete cells of iron to limit growth. Chelators have not proven, however, to be successful anti-cancer agents. As cancer cells also have increased reactive oxygen species (ROS) we hypothesized that a contrarian approach of enhancing iron entry would allow for further increased generation of ROS causing oxidative damage and cell death. Molecules that accelerate iron uptake were identified using high through-put screening of a reporter system of K562 cells loaded with the divalent metal chelator calcein whose fluorescence is quenched with chelation of Fe2+. A small molecule library consisting of ∼11,000 compounds was screened and a class of compounds identified that stimulated iron-induced quenching of calcein fluorescence. The stimulation was verified by dose-dependent 55Fe uptake in various cancer cell lines. The lead compound LS081 exhibited an IC50 = 1.22 ± 0.48 μM and also increased the uptake of Fe from transferrin (Tf). LS081 decreased PC-3 cell growth in the presence of Fe2+ by 64 ± 8% compared to 14 ± 1% decrease with LS081 alone (mean decrease ± SEM, n = 3) with minimal effects on the immortalized, non-malignant prostate cell line 267B1 cells. LS081 plus Fe increased DNA strand breaks and induced cell cycle arrest at the G1 phase of PC-3 cells. In addition, LS081 markedly decreased HIF-1α and HIF-2α levels in the DU-145 prostate cancer and the MDA-MB-231 breast cancer cell lines. LS081 also stimulated ROS production by nearly 3-fold and decreased clonogenicity by greater than 50% in PC-3 cells. In summary, we have developed a high through-put screening technique that identified small molecules that stimulate iron uptake both from ferri-Tf and non-Tf bound iron. These molecules have properties that suggest that these iron facilitator compounds may serve as anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2011-3530

Characterization of an ecto-5′-nucleotidase activity present on the cell surface of Tritrichomonas foetus

Tritrichomonas foetus is the causative agent of sexually transmitted trichomoniasis in cattle. In females, the infection can be associated with infertility, vaginitis, endometritis, abortion or pyometra, leading to significant economic losses in cattle raising. T. foetus is devoid of the ability to synthesize purine nucleotides de novo, depending instead on salvaging purines from the host environment. Ecto-5′-nucleotidase catalyzes the final step of extracellular nucleotide degradation, the hydrolysis of nucleoside 5′-monophosphates to the corresponding nucleosides and Pi. In this work we show that living, intact cells of T. foetus were able to hydrolyze 5′AMP at a rate of 12.57 ± 1.23 nmol Pi × h −1 × 10 −7 cells at pH 7.2 and the 5′AMP hydrolysis is due to a plasma membrane-bound ecto-enzyme activity. The apparent K m for 5′AMP was 0.49 ± 0.06 mM. In addition to 5′AMP, the enzyme hydrolyzed all substrate monophosphates tested except 3′AMP. No divalent metals or metal chelators were able to modulate enzyme activity. Phosphatase inhibitors did not have an effect on ecto-5′-nucleotidase activity while ammonium molybdate did inhibit the activity in a dose dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. These results indicate the existence of an ecto-5′-nucleotidase that may play a role in the salvage of purines.

Topical Metal Chelation Therapy Ameliorates Oxidation-Induced Toxicity in Diabetic Cataract

Oxidative stress plays a critical role in cataractogenesis, the leading cause of blindness worldwide. Since transition metals generate reactive oxygen species (ROS) formation, metal chelation therapy has been proposed for treatment of cataracts. However, the effectiveness of most chelators is limited by low tissue penetrability. This study is the first to demonstrate that the topically applied divalent metal chelator ethylenediamine tetraacetic acid (EDTA) combined with the carrier and permeability enhancer methyl sulfonyl methane (MSM) ameliorates both oxidation-induced lens opacification and the associated toxic accumulation of protein-4-hydroxynonenal (HNE) adducts. Both in vitro (rat lens culture) and in vivo (diabetic rats), EDTA–MSM (1) significantly reduced lens opacification by about 40–50%, (2) significantly diminished lens epithelial cell proliferation and fiber cell swelling in early stages of cataract formation in vivo, and (3) notably decreased the levels of protein–HNE adducts. These findings have important implications specifically for the treatment of cataract and generally for other diseases in which oxidative stress plays a key pathogenic role.

Siderophore Receptor IroN Is an Important Protective Antigen Against Salmonella Infection in Chickens

In iron-limiting environments, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium synthesize and secrete several types of siderophore to trap trivalent ferric ions; these bacteria then express siderophore receptors called iron-regulated outer membrane proteins (IROMPs). In this study, we experimentally reproduced iron-limiting environments using a divalent metal chelator. IroN, one of the IROMPs, was purified by affinity chromatography with an anti-IroN-MAb-immobilized column. Thirty-day-old chickens were immunized intramuscularly with purified IroN from Salmonella Typhimurium mixed with Freund's incomplete adjuvant; the chickens were then challenged intravenously with Salmonella Enteritidis. The mortality rate of immunized chickens was 10%. On the other hand, that of control chickens was 80%. By Western blot analysis, specific IgG antibody responses against IroN of Salmonella Enteritidis were identified in chickens immunized with purified IroN. These results indicate that IroN might be promising as an important vaccine component against Salmonella infection in chickens.