RESUMO
AIM: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. MATERIALS & METHODS: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. RESULTS: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. CONCLUSION: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.
Assuntos
Glicosídeo Hidrolases/metabolismo , Mucinas/metabolismo , Naegleria fowleri/enzimologia , Fatores de Virulência/metabolismo , Animais , Western Blotting , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/efeitos dos fármacos , Humanos , Hidroximercuribenzoatos/farmacologia , Camundongos , Microscopia Confocal , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/metabolismo , Naegleria fowleri/patogenicidade , Polissacarídeo-Liases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.
Assuntos
Glucana 1,4-beta-Glucosidase/metabolismo , Animais , Sítios de Ligação , Etildimetilaminopropil Carbodi-Imida/farmacologia , Glucana 1,4-beta-Glucosidase/antagonistas & inibidores , Glucanos , Glucosídeos/metabolismo , Gafanhotos/enzimologia , Concentração de Íons de Hidrogênio , Hidroximercuribenzoatos/farmacologia , Himecromona/análogos & derivados , Himecromona/metabolismo , Cinética , Masculino , Modelos Químicos , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD(50) values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin - a urease isoform from jackbean seeds that displays a toxic effect in mice (LD(50) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x mL(-1) for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni(+2) and Zn(+2) ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.
Assuntos
Bacillus/enzimologia , Fabaceae/enzimologia , Glycine max/enzimologia , Animais , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hidroximercuribenzoatos/farmacologia , Concentração Inibidora 50 , Insetos , Inseticidas/farmacologia , Cinética , Agregação Plaquetária/efeitos dos fármacos , Fatores de Tempo , Ureia/metabolismo , Urease/metabolismoRESUMO
Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.
Assuntos
Lectinas/química , Lectinas/metabolismo , Phaseolus/enzimologia , Proteínas de Plantas , Toxinas Biológicas , Urease/química , Urease/fisiologia , Sequência de Aminoácidos , Animais , Plaquetas/enzimologia , Cromatografia em Gel , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hemaglutininas/metabolismo , Hidroximercuribenzoatos/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Lectinas de Plantas , Ligação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Ureia/metabolismo , Urease/metabolismo , Zinco/metabolismoRESUMO
To determine whether lysophospholipids mobilize cellular Ca2+, intact rat islets were prelabelled with 45Ca2+ and subjected to three maneuvers designed to simulate the physiologic accumulation of lysophospholipids: (1) exogenous provision; (2) addition of porcine pancreatic phospholipase A2; and (3) provision of p-hydroxymercuribenzoic acid, which impedes both the reacylation and hydrolysis of endogenous lysophospholipids, leading to their accumulation in islets. Each maneuver provoked 45Ca2+ efflux at concentrations nearly identical to those previously reported to induce insulin release in the absence of toxic effects on the islets. Lysophosphatidylcholine (lysoPC) and lysophosphatidylinositol were active, whereas the ethanolamine and serine derivatives, and lysophosphatidic acid, were much less effective. The effects of lysoPC were reversible; they also were reduced by lanthanum or gentamicin (which are probes of superficial, plasma membrane-bound stores of Ca2+) or by prior depletion of membrane-bound cellular Ca2+ stores using ionomycin, but not by removal of extracellular Ca2+ or Na+. The effects of lysoPC, phospholipase A2 and p-hydroxymercuribenzoic acid were largely independent of any hydrolysis to, or accumulation of, free fatty acids as assessed by resistance to dantrolene or trifluoperazine (which selectively reduce arachidonic acid-induced 45Ca2+ efflux and insulin release). Thus, lysophospholipids are a newly recognized class of lipid mediators which may promote insulin release at least in part via mobilization of a pool(s) of Ca2+ ('trigger Ca2+') bound in the plasma membrane and possibly in other cellular membranes.
Assuntos
Cálcio/metabolismo , Ilhotas Pancreáticas/metabolismo , Lisofosfolipídeos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Éteres/farmacologia , Ácidos Graxos não Esterificados/biossíntese , Gentamicinas/farmacologia , Glucose/farmacologia , Hidroximercuribenzoatos/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ionomicina , Ilhotas Pancreáticas/efeitos dos fármacos , Lantânio/farmacologia , Masculino , Fosfolipases A/farmacologia , Fosfolipases A2 , Ratos , Ratos EndogâmicosRESUMO
Susceptibility to inorganic mercuric ions and to organomercurials of 237 Pseudomonas aeruginosa clinical strains isolated in Mexico was determined by agar dilution tests. Resistant strains fell into two classes: i) narrow-spectrum resistant strains (27% of total isolates) resistant only to mercuric ions and to merbromin, and most grouped in pyocin type 1; and ii) broad-spectrum resistant strains (5%) with additional resistances to thimerosal, phenylmercury, methylmercury and p-hydroxymercuribenzoate, that belonged mostly to pyocin type 10. Mercurial resistant isolates showed a higher proportion of resistance to antibiotics and metals than did mercurial sensitive isolates, and broad-spectrum resistant strains had the highest frequency of resistance to antibiotics and to tellurite and arsenate.