Endogenous production of hydrogen sulfide in isolated bovine eye

Hydrogen sulfide (H₂S) is a novel gasotransmitter with physiological and pathological functions in vascular homeostasis, cardiovascular system and central nervous system. In the present study, we determined the endogenous levels of H₂S in various tissues of the bovine eye. We also examined the basal levels of H₂S in response to donors (sodium hydrosulfide, NaHS and sodium sulfide, Na₂S), substrate (l-cysteine), inhibitors (propargylglycine, PAG and aminooxyacetic acid, AOA) and activator (S-adenosyl-l-methionine, SAM) of this gas in the bovine retina. H₂S was measured using a well established spectrophotometric method. The highest concentration of endogenous H₂S was detected in cornea (19 ± 2.85 nmoles/mg protein, n = 6) and retina (17 ± 2.1 nmoles/mg protein, n = 6). Interestingly, H₂S was not present in vitreous humor. The inhibitors of CSE and CBS; PAG (1 mM) and AOA (1 mM), significantly attenuated the production of H₂S in the bovine retina by 56.8 and 42%, respectively. On the other hand the activator of CBS; SAM (100 μM), H₂S donors; NaHS (1 μM) and Na₂S (100 μM), significantly increased endogenous levels of H₂S in bovine retina. l-cysteine (10–300 μM) produced a significant (P < 0.05) concentration-dependent increase in H₂S levels reaching a maximal at 300 μM. We conclude that H₂S is endogenously produced in various tissues of the isolated bovine eye. Moreover, endogenous levels of H₂S are enhanced in the presence of substrate (l-cysteine), an activator of CBS (SAM) and H₂S donors but are blocked by inhibitors of enzymes that synthesize this gas in neural retina.     

Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Effects of crude oil on survival,morphology, and anatomy of two aquatic macrophytes from the Amazon floodplains

Aquatic herbaceous macrophytes grow in profusion in the Amazon fertile varzea floodplains. A large number of species occur but only a few are particularly abundant, supporting food chains, contributing substantially to carbon and nutrient cycles. Their growth and role in the ecosystem depend, among other, on its life cycles and habits, floating or semi-aquatic. Although in the last decades, petroliferous activity intensified in the Central Amazon region and so did oil spills, the effect of petroleum on the native aquatic plants is unknown. The present study was designed to test experimentally the survival and morpho-anatomical modifications of the free floating water hyacinth Eichhornia crassipes and the semi-aquatic grass Echinochloa polystachya to 10 different concentrations of crude oil. Higher concentrations of crude oil caused the mortality in both species; however, lethal dose (LD₅₀) values showed that E. polystachya was more sensitive than E. crassipes. Despite the higher tolerance of E. crassipes, the inhibition of root and leaf growth as well as anatomical modifications in leaves were registered in higher concentrations. Additionally, the oil caused a reduction in leaf numbers in both species. Although mortality of the floating species was lower, it may increases over time, since important alterations in morphology and anatomy occurred. These results show that oil spills in the Amazon varzea can cause severe alterations in the aquatic flora and in the floodplain dynamics.     

Curli fimbria production among Escherichia coli strains isolated from children with diarrhea and healthy children

E. coli curli fimbria are produced during starvation and at room temperature. In the study curli synthesis among E. coli strains isolated from children with diarrhea and healthy children was investigated at human body temperature and in the variable atmosphere conditions: aerobic, supplemented with CO2 and anaerobic. The ability of curli synthesis was estimated using qualitative and quantitative methods basing on Congo red binding by curli. Curli production of all E. coli strains examined was observed at temperature 37 degrees C during anaerobic cultivation, confirming their role in colonization of intestinal mucosa.     

Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas

Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.     

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2′-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.     

Specific characteristics of the strains isolated from a thermoacidophilic microbial community oxidizing antimony sulfide ore

Investigation of the phenotypic properties of three mixotrophic bacteria, strains Sb-K, Sb-F, and Sb-S, isolated from an aboriginal thermoacidophilic microbial community participating in biooxidation of ore with high antimony content (26%) and ore concentrates from the Olympiadinskoe deposit under semicontinuous cultivation conditions at 46 ± 1°C, revealed the differentiating characteristics of these strains. The isolated cultures grew lithotrophically through different numbers of transfers: strains Sb-F and Sb-K grew through seven and eight transfers, respectively, and strain Sb-S grew through two or three transfers. Strains Sb-K and Sb-S utilized a wide range of organic substrates for active organotrophic growth during nine or ten transfers, while strain Sb-F was less tolerant to organic compounds. Strain Sb-K grew on a medium with the ore and sulfide ore concentrates in the pH range of 1.0–3.0. Growth of strains Sb-F and Sb-S occurred in the pH ranges of 1.0–2.5 and 1.5–5.5 on media with Fe²⁺ and S⁰, respectively.. The optimal initial pH values of the media, corresponding to the maximum specific growth rates, were 1.6–1.7, 1.9, and 2.0–3.0 for strains Sb-K, Sb-F, and Sb-S, respectively. All three strains were able to grow within a broad temperature range, 20–65°C, with an optimum at 46°C (Sb-K), 40–46°C (Sb-F), and 48–50°C (Sb-S). According to the results of DNA-DNA hybridization and phylogenetic analysis, as well as their phenotypic characteristics, the isolates can be classified as novel strains of species of the genus Sulfobacillus. Strains Sb-K, Sb-F, and Sb-S, isolated as predominant cultures on the media with sulfide compounds, iron, or sulfur, respectively, were affiliated to the species S. thermotolerans, S. sibiricus, and S. thermosulfidooxidans.     

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetics and telemetry indicate unexpected movements among structured populations for Brachyplatystoma platynemum in the Amazon

The genetic analysis of Brachyplatystoma platynemum individuals sampled from the lower Madeira River reinforces the existence of two structured populations in the Amazon Basin (Madeira and Amazon populations). However, the recapture of an individual from the Amazon population in the Solimões River, which was telemetry-tagged in the Madeira River after the damming, indicates that fish from the Amazon population move between the two river systems. This has not yet been observed, however, in the Madeira River population, which is currently divided and isolated in the lower and upper Madeira River by the construction of two dams.     

Studies of two Frankia strains isolated from Trevoa trinervis Miers

The Frankia strains TtI 11 and TtI 12 isolated from T. trinervis Miers were characterized regarding their carbon source utilization, intrinsic antibiotic resistance, infectivity, and effectivity on the original host. Both strains grew on BAP medium supplemented with glucose, maltose, and sucrose, but differed in their ability to use other carbon sources such as propionate, pyruvate, acetate, succinate, citrate, and mannitol. The isolates were sensitive to five of the twelve antibiotics tested at 1 μg mL⁻¹ concentration: chloramphenicol, tobramycin, eritromycin, streptomycin, and rifampicin. They exhibited a variable degree of resistance at 1 μg mL⁻¹ concentraction to penicillin G, 4-fluorouracil, oleandomycin, and lincomycin. Both isolates were able to infect and nodulate the original host plant, and thus represent the first reported infective and effective microsymbionts for T. trinervis Miers, a rhamnaceous actinorhizal host. R O D Dixon Section editor     

Biofilm production by clinical strains of Acinetobacter baumannii isolated from patients hospitalized in two tertiary care hospitals

Microbial biofilms are considered as virulence factors. During the present study, 34 clinical strains of Acinetobacter baumannii, isolated from patients hospitalized in two tertiary care hospitals, were examined for biofilm formation. These strains showed high variability in biofilm formation. Furthermore, no relation could be found between the ability of biofilm production and molecular type, carbapenem resistance, site of isolation of the clinical strains of A. baumannii and disease severity. Interestingly, in two cases an increase in biofilm formation could be detected in A. baumannii isolates cultured from the same patient upon prolonged hospitalization.     

Stable nitrogen isotope ratios of macrophytes and associated periphyton along a nitrate gradient in two subtropical, spring-fed streams

1. An increase in human population and associated changes in land use have caused an increase in groundwater nitrate concentrations throughout central Florida. Within the region, this nitrate‐laden groundwater returns to the surface via numerous large springs that serve as the origin of flow for many coastal streams and rivers. These rivers can exhibit strong nitrate gradients because of the high nutrient uptake potential of the rivers. 2. We hypothesised that downstream declines in nitrate concentrations would be manifested spatially as increases in the δ¹⁵N of the residual pool of nitrate, macrophytes and periphyton as a consequence of isotopic fractionation associated with preferential use of ¹⁴NO₃⁻. This hypothesis was tested in two spring‐fed river systems, the Chassahowitzka and Homosassa rivers, along Florida's central Gulf of Mexico coast. 3. In general, δ¹⁵N values of nitrate, macrophytes and periphyton increased with decreasing fraction of nitrate remaining in each of the two study systems. The fractionation associated with nitrate uptake by macrophytes and associated periphyton was determined from the relationship between δ¹⁵N of both constituents of the macrophyte community and the fraction of nitrate removed from the system. Values for fractionation by macrophytes and periphyton ranged from 1.9‰ to 3.6‰ and from 0.7‰ to 2.5‰, respectively.     

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 μg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.     

The response of two submerged macrophytes and periphyton to elevated temperatures in the presence and absence of snails: a microcosm approach

Global warming may affect snail–periphyton–macrophyte relationships in lakes with implications also for water clarity. We conducted a 40-day aquaria experiment to elucidate the response of submerged macrophytes and periphyton on real and artificial plants to elevated temperatures (3°C) under eutrophic conditions, with and without snails present. With snails, the biomass and length of Vallisneria spinulosa leaves increased more at the high temperature, and at both temperatures growth was higher than in absence of snails. The biomass of periphyton on V. spinulosa as well as on artificial plants was higher at the highest temperature in the absence but not in the presence of snails. The biomass of Potamogeton crispus (in a decaying state) declined in all treatments and was not affected by temperature or snails. While total snail biomass did not differ between temperatures, lower abundance of adults (size >1 cm) was observed at the high temperatures. We conclude that the effect of elevated temperature on the snail–periphyton–macrophyte relationship in summer differs among macrophyte species in active growth or senescent species in subtropical lakes and that snails, when abundant, improve the chances of maintaining actively growing macrophytes under eutrophic conditions, and more so in a warmer future with potentially denser growth of periphyton.     

VISA and hetero-VISA among Staphylococcus aureus strains isolated from clinical cases

The VISA (vancomycin intermediate Staphylococcus aureus) and hetero-VISA strains were found among all isolates of S. aureus obtained in the years 1997-2000. The frequency of VISA was 0.3% and h-VISA was about 3%. Most but not all of the h-VISA and all VISA strains were methicillin resistant. Moreover the usefulness of different methods enabling recognition of the h-VISA strains was compared.     

Cyanotoxin production and phylogeny of benthic cyanobacterial strains isolated from the northeast of Brazil

Most of the knowledge about cyanobacteria toxin production is traditionally associated with planktonic cyanobacterial blooms. However, some studies have been showing that benthic cyanobacteria can produce cyanotoxins. According to this, we aimed to evaluate the production of microcystins and saxitoxins in benthic cyanobacteria isolated from aquatic ecosystems in the Northeast of Brazil and to use a polyphasic approach for their identification. Forty-five clonal strains were isolated from rivers and water supply reservoirs, and identified using morphological and molecular phylogenetic characteristics. In order to evaluate the toxins production, the strains were screened for genes involved in the biosynthesis of microcystins and saxitoxins, positive results were confirmed and cyanotoxins quantified using HPLC. Eight species were identified belonging to the Phormidiaceae, Pseudanabaenaceae and Nostocaceae families. This is the first study in Brazil that shows that strains from the Geitlerinema genus correspond to at least three phylogenetic lineages, which possibly correspond to three distinct species to be subsequently reclassified. The strains that showed one of the genes involved in the cyanotoxins production were analyzed by HPLC and Geitlerinema amphibium, Geitlerinema lemmermannii, Cylindrospermum stagnale and Phormidium uncinatum were identified as producing one or more saxitoxins variants. Thus, this is the first report of saxitoxins production for those first three species and the first report in Brazil for P. uncinatum.     

Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains

We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (ΔldhA, ΔfrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h⁻¹ g⁻¹ dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h⁻¹ l⁻¹ (20.2 l h⁻¹ l⁻¹ at 37 °C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications.