In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms.

Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage.

Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism.

Characterization of a human-pathogenic Acanthamoeba griffini isolated from a contact lens-wearing keratitis patient in Spain.

Amoebae were isolated from contact lenses of a symptomatic lens wearer in Spain. Protozoa were characterized by studying their morphology, biology, protease activity and the 18S rRNA gene sequence. Morphology of the organism was observed by light microscopy, scanning electron microscopy and transmission electron microscopy. Its structure corresponded to an amphizoic amoeba. The protozoa grew well at 37 °C and poorly at lower temperatures. In addition, it was capable of lysing mammalian cells in vitro. A major 56 kDa proteolytic enzyme was observed in amoeba crude extracts by gelatin-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Most proteolytic enzymes in protozoa extracts showed significant activity over a wide range of pH (3-9) and temperature (8-45 °C) values. The assays on inhibition of protease activity indicated strongly that enzymes detected in amoeba extracts corresponded to serine proteases and, to a lesser extent, cysteine proteases. The use of proteinase inhibitors on a tissue culture model proved that the proteinase activity is critical for developing focal lesions in HeLa cell monolayers. Finally, partial sequencing of the 18S ribosomal RNA gene and phylogenetic analyses indicated that the isolate is closely related to Acanthamoeba griffini H37 from the UK (T3 genotype).

In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites.

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

In vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against Acanthamoeba castellanii UAH-T17c3 trophozoites.

Acanthamoeba is one of the most common free-living amoebas which is widespread in the environment and can infect humans, causing diseases such as keratitis and encephalitis. In this paper we examine for the first time the amebicidal activity of the family of cationic dendrimers nG-[Si{(CH(2))(3)N(+)(Me)(Et)(CH(2))(2)NMe(3) (+)}2I(-)]( x ) (where n denotes the generations: zero (n = 0, x = 1), first (n = 1, x = 4), and second (n = 2, x = 8); for simplicity, they were named as 0G-CNN2, 1G-CNN8, and 2G-CNN16, respectively) against Acanthamoeba castellanii UAH-T17c3 trophozoites. In order to test the amebicidal activity, we cultured the strain A. castellanii UAH-T17c3 in PYG-Bactocasitone medium and later, we treated it with different concentrations of these dendrimers and monitored the effects and damage by optical count, flow cytometry, and scanning electron microscopy. The results showed that all the nanosystems assayed had a strong amebicidal activity. The dendrimer 1G-CNN8 was the most effective against the amoeba. In the morphology of treated throphozoites of A. castellanii UAH-T17c3 analyzed by light and scanning electron microscopy techniques, morphological changes were evident in amoeba cells, such as loss of pseudopodia, ectoplasm increase, roundness, and cellular lysis. Furthermore, flow cytometry results showed alterations in cell granularity, which was dose-time dependent. In conclusion, this family of cationic carbosilane dendrimers has a strong amebicidal activity against the trophozoites of A. castellanii UAH-T17c3 in vitro. They could potentially become new agents significant to the development of new amebicidal compounds for prevention and therapy of Acanthamoeba infections.

Acanthamoeba castellanii: in vitro UAH-T17c3 trophozoite growth study in different culture media.

Acanthamoeba is one of the most common free-living amoebae. It is widespread in the environment and can infect humans, causing diseases such as keratitis and encephalitis. In this study, we used a strain of Acanthamoeba castellanii (UAH-T17c3) isolated from cooling towers, and we evaluated the efficiency of three different culture media in its growth, with the aim of selecting one which allowed better growth, was easier to prepare, and was able to keep the trophozoites by long periods of time. We compared the growth of A. castellanii in peptone-yeast extract-glucose (PYG, the most commonly used medium to grow this strain) to the growth in PYG-Bactocasitone (PYG with 2% Bactocasitone) and brain-heart infusion broth (BHI is a standard microbiological medium rarely used in the culture of amoebae). Flow cytometry and cell count results showed all three media allowed the growth of trophozoites. PYG-Bactocasitone was shown to be the best for long-term culture. The BHI and PYG-Bactocasitone media have not been used for Acanthamoeba spp. trophozoite growth. In view of the results, we can affirm that these media are adequate to grow the above-mentioned strain for in vitro screening assays.

Microbiological water quality and its relation to nitrogen and phosphorus at the Pareja limno-reservoir (Guadalajara, Spain).

Bordering on the edge of the Entrepeñas reservoir (Guadalajara, Spain), next to the village of Pareja, a small dam that allows a body of water to develop with a constant level has been built. Initiatives like this (which we have termed "limno-reservoirs") are innovative in Spain and around the world. Earlier reservoirs such as this one were constructed to create a habitat for birds, but the Pareja limno-reservoir is the first to promote socio-economic development. In order to study this limno-reservoir, this research group set up an environmental observatory, analyzing, among other variables, microbiological water quality and nutrient content. After a year and a half of research, it was observed that the concentration of microorganisms is lower in the limno-reservoir than in the river that feeds it, possibly due to the nutrient depletion in the lentic ecosystem. In the limno-reservoir, the total coliforms and enterococci concentrations fall within the European Bathing Water Directive limits, but in the river these concentrations are sometimes higher. The nutrient load in the limno-reservoir is low, with nutrient variations influencing native microorganisms, but not for total coliforms and enterococci. However, the development of special conditions in the bottom has been observed in winter, facilitating coliforms and enterococci survival. This research is very interesting since the creation of limno-reservoirs is rising in Spain and no research is being done on their behaviour.

Release of ferulic acid and feruloylated oligosaccharides from sugar beet pulp by Streptomyces tendae.

Given several promising industrial applications of ferulic acid, this study was designed to identify actinomycete strains able to release high levels of this acid from sugar beet pulp (SBP). Out of 47 strains tested, 37% were found to release free ferulic acid from the growth substrate. One strain, identified as Streptomyces tendae by 16S RNA gene sequencing, was capable of releasing 80% of the ferulic acid ester-linked to the pectin in SBP after 5 days of growth. These data suggest that some actinomycetes are able to release ferulic acid and feruloylated oligosaccharides from SBP. During growth on SBP, it seems that Streptomyces species solubilize and release feruloylated oligosaccharides by specific carbohydrase activities before de-esterification and release of free ferulic acid.

xln23 from Streptomyces chattanoogensis UAH23 encodes a putative enzyme with separate xylanase and arabinofuranosidase catalytic domains.

The xylanase gene xysA of Streptomyces halstedii JM8 was used to isolate a DNA fragment from a gene library of Pstl-digested chromosomal DNA of the lignocellulolytic actinomycete Streptomyces chattanoogensis CECT-3336. Nucleotide sequence analysis revealed a gene (xln23) encoding a bifunctional multimodular enzyme bearing two independent xylanase and alpha-L-arabinofuranosidase domains separated by a Ser/Gly-rich linker. The N terminus of the predicted protein showed high homology to family F xylanases. The C terminus was homologous to amino acid sequences found in enzymes included in the glycosyl hydrolase family 62 and, in particular, to those of alpha-L-arabinofuranosidase AbsB from Streptomyces lividans. PCR and RT-PCR experiments showed that the nucleotide sequences corresponding to each domain are arranged as expected on the chromosomal DNA and that they are cotranscribed. To our knowledge, this is the first description of xylanase and arabinofuranosidase domains in a same open reading frame.

Purification and properties of a chitinase from Penicillium oxalicum autolysates.

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54,900 as estimated from SDS gel electrophoresis and 21,500 from gel filtration. The optimum pH and temperature were 5.0 and 35 degrees C, respectively. The enzyme was stable at temperatures up to 45 degrees C and in a pH range between 4.0 and 6.0. The Km was 2.5 mg ml-1 for colloidal chitin, Hg2+ and Ag+ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.

Phanerochaete chrysosporium and its natural substrate.

We seek to define more fully how Phanerochaete chrysosporium degrades its natural substrate, lignocellulose. This contribution concerns several relevant topics. Mineralisation of [14C]DHP, as a model for lignin degradation, showed that a set of genetically defined meiotically derived products of strain ME446 differed in their degradative ability and also that, under optimum conditions for mineralisation, extracellular lignin peroxidase activity was absent. Xylanolytic and xylosidase/beta(1-->3) glucanase activities are also described. The complexity of the CBHI gene family is described and differential splicing of a CBHI gene transcript is proposed. In contrast to the multiplicity of CHBI genes there is a single CBHII gene. PCR methods were developed to analyse differential gene expression on different substrates. We have also developed a transformation system involving a reporter construct for the analysis of CBHI promoter function.

A Phanerochaete chrysosporium beta-D-glucosidase/beta-D-xylosidase with specificity for (1-->3)-beta-D-glucan linkages.

Phanerochaete chrysosporium is the best studied organism with respect to lignin degradation, but its degradation of the xylan component of lignocellulose is only now being studied. When grown on oat spelt xylan (mainly arabinoxylan), it produces an enzyme with beta-D-xylosidase and beta-D-glucosidase activity. This enzyme was purified by ultrafiltration followed by ammonium sulphate precipitation, anion-exchange chromatography using DEAE Biogel and Mono Q, and gel filtration using Superose 12. It is extracellular, with an apparent M(r) value of 44,500 as determined by SDS-PAGE; the pI is 4.67 and activity is maximal at pH 5 and 60 degrees C. The enzyme is of particular interest because its principal activity is against laminaribiose (3-O-beta-D-glucopyranosyl-D-glucopyranose and laminarin [(1-->3)-beta-D-glucan with ca. 3% of beta-(1-->6) branches] rather than cellobiose and xylobiose. It was competitively inhibited by D-glucono-1,5-lactone and deoxynojirimycin; with p-nitrophenyl beta-D-xylopyranoside as substrate, the Ki values were 32 and 87.5 microM, respectively, and with p-nitrophenyl beta-D-glucopyranoside, they were 35 and 68.7 microM, respectively. The Km values with p-nitrophenyl beta-D-xylopyranoside and p-nitrophenyl beta-D-glucopyranoside as substrates were 3.51 and 5.30 mM, respectively.

Polarimetry and 13C n.m.r. show that the hydrolyses of beta-D-glucopyranosyl fluoride by beta(1-->3)-glucanases from Phanerochaete chrysosporium and Sporotrichum dimorphosporum have opposite stereochemistries.

The time courses of optical rotation and fluoride ion release during hydrolysis of beta-D-glucopyranosyl fluoride by the beta(1-->3)-glucanase of Phanerochaete chrysosporium (J. L. Copa-Patiño and P. Broda, unpublished work) indicated that the initial sugar product was beta-D-glucopyranose. This was confirmed by monitoring the hydrolysis of 1-[13C]beta-D-glucopyranosyl fluoride by this enzyme with 13C n.m.r. (without proton decoupling). The same two techniques were used to confirm that hydrolysis of beta-D-glucopyranosyl fluoride by the exo beta(-->3)-glucanase of 'Basidiomycete QM 806' (identified as Sporotrichum dimorphosporum) yielded alpha-glucopyranose as first sugar product, in accordance with previous results using laminarin as substrate [Parrish and Reese (1963) Carbohydr. Res. 3, 424-429; Nelson (1970) J. Biol. Chem. 245, 869-872].

Effect of beta-glucanases on Penicillium oxalicum cell wall fractions.

The polysaccharidic effect of a purified 1,3-beta-glucanase, a purified beta-glucosidase, and of partially purified endo-1,3-beta-glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied. Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F1, an alpha-glucan; F3, a beta-glucan; F4, a chitin-glucan; and F4b, a beta-glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation. The enzymes were found to degrade fraction F4b (beta-glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.

Cephalosporin C acylase in the autolysis of filamentous fungi.

Cephalosporin C acylase activity was studied using fluorescamine determination of free--NH2 groups produced in the deacylation of cephalosporin C by the enzyme. Fourteen fungi from different genera were studied and low extracellular cephalosporin C acylase activity was found in the genera Aspergillus, Fusarium and Penicillium. Forty one fungi of these genera were checked but not all presented acylase activity. The enzyme was generally found to be an extracellular enzyme and during the process of autolysis its activity increased with incubation time and with increasing pH of the medium. In no case was beta-lactamase activity detected. Penicillium rugulosum and Penicillium griseofulvum were identified as good cephalosporin C acylase producers. Deacetyl esterase activity was also detected in these fungi.

Purification and properties of a beta-glucosidase from Penicillium oxalicum autolysates.

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.

Purification and properties of a 1,3-beta-glucanase from Penicillium oxalicum autolysates.

High 1,3-beta-glucanase activity was detected during autolysis in a culture medium containing Penicillium oxalicum. It was due to the combined action of four enzymes. The purification process for the major enzyme produced a homogeneous band in the SDS polyacrylamide gel that corresponded to a molecular weight of 79,400 daltons. The enzyme pI was 6.3 and it was only active against 1,3-beta-glucans, with a S0.5 of 0.23 mg ml-1 against laminarin. The enzymatic optima were found at pH 4 and 55 degrees C, and instability was evident when pH and temperature were altered. The enzyme was not active against oxidated laminarin and was barely inhibited by glucono-D-lactone. Hg2+, Ag+ and Fe2+ were effective inhibitors. The enzyme was adsorbed by concanavalin-A-sepharose.