Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either P-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CATactivities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was failing from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with P-glucan had a higher survival rate (60%) than the group not pre-treated with P-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses. (C) 2009 Elsevier Ltd. All rights reserved.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp PCC 7120 (Cyanobacterium) under simulated microgravity

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity. (C) 2004 Elsevier Ltd. All rights reserved.

Effect of condensed tannins from tropical legumes on the activity of fibrolytic enzymes from the rumen fungus Neocallimastyx hurleyensis

A study was conducted to assess the effect of condensed tannins on the activity of fibrolytic enzymes from the anaerobic rumen fungus, Neocallimastix hurleyensis and a recombinant ferulic acid esterase (FAE) from the aerobic fungus Aspergillus niger. Condensed tannins were extracted from the tropical legumes Desmodium ovalifolium, Flemingia macrophylla, Leucaena leticocephala, Leucaena pallida, Calliandra calothyrsus and Clitoria fairchildiana and incubated in fungal enzyme mixtures or with the recombinant FAE. In most cases, the greatest reductions in enzyme activities were observed with tannins purified from D. ovalifolium and F macrophylla and the least with tannins from L leucocephala. Thus, whereas 40 mu g ml(-1) of condensed tannins from C. calothyrsus and L. leucocephala were needed to halve the activity of N. hurleyensis carboxymethylcellulase (CMCase), just 5.5 mu g ml(-1) of the same tannins were required to inhibit 50% of xylanase activity. The beta-D-glucosidase and beta-D-Xylosidase enzymes were less sensitive to tannin inhibition and concentrations greater than 100 mu g ml(-1) were required to reduce their activity by 50%. In other assays, the inhibitory effect of condensed tannins when added to incubation mixtures containing particulate substrates (the primary cell walls of E arundinacea) or when bound to these substrate was compared. Substrate-associated tannins were more effective in preventing fibrolytic activities than tannins added directly to incubations solutions. It was concluded that condensed tannins from tropical legumes can inhibit fibrolytic enzyme activities, although the extent of the effect was dependent on the tannin, the nature of its association with the substrate and the enzyme involved. (c) 2005 Elsevier Inc. All rights reserved.

Screening for pectinolytic activity of wood-rotting basidiomycetes and characterization of the enzymes

Seventy-five fungal strains from different groups of basidiomycetes, newly isolated from rotten wood, were screened for pectinolytic activity. Despite the fact that basidiomycetes are scarcely referred to as pectinase producers, the polygalacturonase (PG) activity was detected in 76 % of the strains; 16 % with activity higher than 40 nkat/g, 40 % between 13.3 and 40 nkat/g, and 44 % with activity lower than 13.3 nkat/g. The highest productions were obtained among the fungi from order Aphyllophorales, family Polyporaceae. The characterization of the enzymes from the highest PG producers (Lentinus sp., Gloeophyllum striatum, Pycnoporus sanguineus, Schizophyllum commune) showed optimum temperature for catalytic activity at 60-70°C and two peaks of pH optimum (3.5-4.5 and 8.5-9.5). The enzymes exhibited high pH stability (3.0-11.0) but after incubation at 40°C for 1 h their activity dropped by 18-73 %.

Serum activity of creatine kinase and aminotransferase aspartate enzymes of horses submitted to muscle biopsy and incremental jump test

The objective was to evaluate serum activity of the enzymes creatine kinase (CK) and aspartate aminotransferase (AST), which are leakage enzymes responsive to muscle injury, of athletic horses that underwent muscle biopsy and incremental jump test (IJT) involving incremental jumps. The animals were grouped as follows: the first group, horses with history of superior performance (SP); the second, with a history of inferior performance (IP); and lastly, a control group (CG). All groups underwent biopsy of the gluteus medius muscle, while groups SP and IP were also submitted to the incremental jump test (IJT) 24 hours after biopsy. The IJT consisted of three stages with 40 jumps each, where jump height increased progressively, from 40 to 60 and last, 80cm. Blood samples were drawn before biopsy, and 6 and 24 hours after the exercise as well. The levels of CK serum activity increased 6 hours after exercise and decreased 24 hours later in all groups, including CG. AST activity did not increase after biopsy and exercise. There was no increase of both enzyme activities that could be attributed to the exercise, possibly due to exercise short duration and/or low intensity. We conclude that the muscle biopsy was able to show that there was enough stimulus to cause CK enzyme leakage into the plasma, and consequent detection of increased serum activity, while the incremental jump test did not.

Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle

The effect of short-term creatine (Cr) supplementation upon content of skeletal muscle-derived-reactive oxygen species (ROS) was investigated. Wistar rats were supplemented with Cr (5 g/kg BW) or vehicle, by gavage, for 6 days. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated for evaluation of ROS content using Amplex-UltraRed reagent. The analysis of expression and activity of antioxidant enzymes (superoxide dismutase 1 and 2, catalase and glutathione peroxidase) were performed. Direct scavenger action of Cr on superoxide radical and hydrogen peroxide was also investigated. Short-term Cr supplementation attenuated ROS content in both soleus and EDL muscles (by 41 and 33.7%, respectively). Cr supplementation did not change expression and activity of antioxidant enzymes. Basal TBARS content was not altered by Cr supplementation. In cell-free experiments, Cr showed a scavenger effect on superoxide radical in concentrations of 20 and 40 mM, but not on hydrogen peroxide. These results indicate that Cr supplementation decreases ROS content in skeletal muscle possibly due to a direct action of Cr molecule on superoxide radical.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.

Activity of ETS enzymes and phosphatase in the Sea of Okhotsk and Kuril region in summer 1992 and 1993

An estimate of rate of transformation of organic matter and regeneration of nutrients (in particular phosphorus) was calculated for different regions of the Sea of Okhotsk. The rate was estimated by means of rate of complete oxidation of organic matter to CO2 and H2O catalyzed by enzymes of the electron transport system (ETS) and rate of hydrolytic splitting of phosphate from organic phosphorus compounds catalyzed by alkaline phosphatase. Organic matter destruction rate was at its maximum on the shelf of Kamchatka and Sakhalin, as well as in the layer of maximum oxygen gradients in deep waters. It was found that zones of intensive primary production were characterized by high rates of phosphorus regeneration, which provided for 80% of primary production when concentration of mineral phosphorus was low.

Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and β-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-tyrosine) and lipid peroxidation (malondialdehyde) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.

Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes

The neurosteroid 3α-hydroxysteroid-5α-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of γ-aminobutyric acid at γ-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5α-dihydroprogesterone, mediated by 5α-reductase, and by reduction to allopregnanolone, mediated by 3α-hydroxysteroid dehydrogenase (3α-HSD). Previous reports suggested that some selective serotonin reuptake inhibitors (SSRIs) could alter concentrations of allopregnanolone in human cerebral spinal fluid and in rat brain sections. We determined whether SSRIs directly altered the activities of either 5α-reductase or 3α-HSD, using an in vitro system containing purified recombinant proteins. Although rats appear to express a single 3α-HSD isoform, the human brain contains several isoforms of this enzyme, including a new isoform we cloned from human fetal brains. Our results indicate that the SSRIs fluoxetine, sertraline, and paroxetine decrease the Km of the conversion of 5α-dihydroprogesterone to allopregnanolone by human 3α-HSD type III 10- to 30-fold. Only sertraline inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3α- and 3α, 17β-reduced or -oxidized androgens mediated by 3α-HSD type IIBrain. Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3α-HSD type IIBrain and 3α-HSD type III mRNAs suggest that SSRIs will affect neurosteroid production in a region-specific manner. Our results may thus help explain the rapid alleviation of the anxiety and dysphoria associated with late luteal phase dysphoria disorder and major unipolar depression by these SSRIs.

The impact of spectral composition and light periodicity on the activity of two antioxidant enzymes (SOD and CAT) in the coral Favia favus

In oligotrophic waters the light spectrum is mostly blue, and therefore the physiological and biochemical responses to blue light occurring in the coral tissue and in the symbiotic algae are important. Examination of the wavelength dependence of two free radical scavenger enzyme activity revealed an increase in activity in the blue light range (440-480 nm) compared to the red (640680 nm) in the full visible light (400-700 nm) range. These data show for the first time the relationship between the action spectra of photosynthesis and the activity of two main antioxidant enzymes in the symbiotic coral Favia favus. It was found that in the animal (host) the enzyme response to the spectral distribution of light was higher than that of the zooxanthellae, probably due to accumulation of free radicals within the host tissue. Furthermore, we found that the activity of these enzymes is affected in nature by the length of the day and night, and in the laboratory, by the duration of the illumination. Changes in the pigment concentrations were also observed in response to growth under the blue region and the whole PAR spectrum, while fluorescence measurements with the fast repetition rate fluorometer (FRRF) showed a decrease in the sigma cross section and a decrease in the quantum yield also in the blue part of the spectrum. These changes of scavenger enzymes activity, pigment concentration and fluorescence yield at different light spectra are vital in acclimatization and survival of corals in shallow water environments with high light radiation. (c) 2005 Elsevier B.V. All rights reserved.