Phytochemical reinvestigation of labdane-type diterpenes and their cytotoxicity from the rhizomes of Hedychium coronarium

A comprehensive reinvestigation of chemical constituents from the rhizomes of Hedychium coronarium resulted in the isolation of one new labdane-type diterpene, together with eight known compounds. Their structures were established by spectroscopic methods. Some of the isolated compounds showed significant cytotoxicity with IC₅₀ values lower than 4 μg ml^?1.     

Optimization of Cd2+ removal by the cyanobacterium Synechocystis pevalekii using the response surface methodology

Response surface methodology (RSM) has been used to optimize the critical parameters responsible for higher Cd²⁺ removal by a unicellular cyanobacterium Synechocystis pevalekii. A three-level Box–Behnken factorial design was used to optimize pH, biomass and metal concentration for Cd²⁺ removal. A coefficient of determination (R²) value (0.99), model F-value (86.40) and its low p-value (F < 0.0001) along with lower value of coefficient of variation (5.61%) indicated the fitness of response surface quadratic model during the present study. At optimum pH (6.48), biomass concentration (0.25 mg protein ml^?1) and metal concentration (5 μg ml^?1) the model predicted 4.29 μg ml^?1 Cd²⁺ removal and experimentally, 4.27 μg ml^?1 Cd²⁺ removal was obtained.     

Direct in vivo interaction of the antibiotic primycin with the plasma membrane of Candida albicans: An EPR study

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg⁺ and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64 μg ml^?1 for 33erg⁺ and 128 μg ml^?1 for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg⁺ and its mutant erg-2 were 12.5 °C and 11 °C, respectively. After 128 μg ml^?1 primycin treatment, these values increased to 17.5 °C and 16 °C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg⁺ and erg-2 were 60 ns and 100 ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8 μs and 1 μs. The results indicate the plasma membrane “rigidizing” effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.     

Synthesis and antimicrobial activity of novel amphiphilic aromatic amino alcohols

We report in this work the preparation and in vitro antimicrobial evaluation of novel amphiphilic aromatic amino alcohols synthesized by reductive amination of 4-alkyloxybenzaldehyde with 2-amino-2-hydroxymethyl-propane-1,3-diol. The antibacterial activity was determined against four standard strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and 21 clinical isolates of methicillin-resistant Staphylococcus aureus. The antifungal activity was evaluated against four yeast (Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis). The results obtained showed a strong positive correlation between the lipophilicity and the antibiotic activity of the tested compounds. The best activities were obtained against the Gram-positive bacteria (MIC = 2–16 μg ml^?1) for the five compounds bearing longer alkyl chains (4c–g; 8–14 carbons), which were also the most active against Candida (MIC = 2–64 μg ml^?1). Compound 4e exhibited the highest levels of inhibitory activity (MIC = 2–16 μg ml^?1) against clinical isolates of MRSA. A concentration of twice the MIC resulted in bactericidal activity of 4d against 19 of the 21 clinical isolates.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Effect of Pichia membranaefaciens in combination with salicylic acid on postharvest blue and green mold decay in citrus fruits

The separate or combined effects of Pichia membranaefaciens and salicylic acid (SA) on the control of blue and green mold decay in citrus fruits were investigated. Results indicate that combining P. membranaefaciens (1 × 10⁸ CFU ml⁻¹) with SA (10 μg ml⁻¹) either in a point-inoculated or dipped treatment provided a more effective control of blue and green mold than separately applying yeast or SA. SA (10 μg ml⁻¹) did not significantly affect P. membranaefaciens growth in vitro but slightly increased the yeast population in fruit wounds. P. membranaefaciens plus SA effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. The combined treatment did not impair quality parameters such as weight loss or titratable acidity, but resulted in low average natural infection incidence and increased total soluble solids and ascorbic acid contents in citrus fruits after 14 d at 20 °C.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Efficacy of biogenic selenium nanoparticles against Leishmania major: In vitro and in vivo studies

Project: This study investigated the in vitro and in vivo effectiveness of biogenic selenium nanoparticles (Se NPs), biosynthesized by Bacillus sp. MSh-1, against Leishmania major (MRHO/IR/75/ER). Procedure: The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity effects of the biogenic Se NPs against both promastigote and amastigote forms of L. major. In a separate in vivo experiment, we also determined the preventive and therapeutic effects of biogenic Se NPs in BALB/c mice following subcutaneous infected with L. major. Results: The MTT assays showed that the highest toxicity occurred after 72 h against both promastigote and amastigote forms of L. major. The cytotoxicity of Se NPs was higher at all incubation times (24, 48, and 72 h) against the promastigote than the amastigote form (p < 0.05). The 50% inhibitory concentrations (IC₅₀) of the Se NPs were 1.62 ± 0.6 and 4.4 ± 0.6 μg ml^?1 against the promastigote and amastigote forms, respectively, after a 72-h incubation period. Apoptosis assays showed DNA fragmentation in promastigotes treated with Se NPs. In an animal challenge, prophylactic doses of biogenic Se NPs delayed the development of localized cutaneous lesions. Moreover, daily administration of Se NPs (5 or 10 mg kg^?1 day^?1) in similarly infected BALB/c mice that had not received prophylactic doses of Se NPs also abolished the localized lesions after 14 days. Conclusion: Based on these in vitro and in vivo studies, biogenic Se NPs can be considered as a novel therapeutic agent for treatment of the localized lesions typical of cutaneous leishmaniasis.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

Continuous Cr(VI) removal by Scenedesmus incrassatulus in an airlift photobioreactor

Cr(VI) removal by Scenedesmus incrassatulus was characterized in a continuous culture system using a split-cylinder internal-loop airlift photobioreactor fed continuously with a synthetic effluent containing 1.0 mg Cr(VI) l^?1 at dilution rate (D) of 0.3 d^?1. At steady state, there was a small increase (6%) on the dry biomass (DB) concentration of Cr(VI)-treated cultures compared with the control culture. 1.0 mg Cr(VI) l^?1 reduced the photosynthetic pigments content and altered the cellular morphology, the gain in dry weight was not affected. At steady state, Cr(VI) removal efficiency was 43.5 ± 1.0% and Cr(VI) uptake was 1.7 ± 0.1 mg Cr(VI) g^?1 DB. The system reached a specific metal removal rate of 458 μg Cr(VI) g^?1 DB d^?1, and a volumetric removal rate of 132 μg Cr(VI) l^?1 d^?1.     

Immune responses,ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254

The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 μg l^?1) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P < 0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 μg l^?1 and bacteriolytic activity 0.5, 5, 50 μg l^?1 treatments decreased significantly, while the proportion of hyalinocytes and the production of O₂^- in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6–15 d. The bacteriolytic activity in 0.05 μg l^?1 treatments, LMS in 0.05, 0.5 μg l^?1 groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9–15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P < 0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.     

Continuous production of selenomethionine-enriched Chlorella sorokiniana biomass in a photobioreactor

This article describes the enrichment of the fresh-water green microalga Chlorella sorokiniana in selenomethionine (SeMet). The microalga was cultivated in a 2.2 L glass-vessel photobioreactor, in a culture medium supplemented with selenate (SeO₄^2?) concentrations ranging from 5 to 50 mg L^?1. Although selenate exposure lowered culture viability, C. sorokiniana grew well at all tested selenate concentrations, however cultures supplemented with 50 mg L^?1 selenate did not remain stable at steady state. A suitable selenate concentration in fresh culture medium for continuous operation was determined, which allowed stable long-term cultivation at steady state and maximal SeMet productivity. In order to do that, the effect of dilution rate on biomass productivity, viability and SeMet content of C. sorokiniana at several selenate concentrations were determined in the photobioreactor. A maximal SeMet productivity of 21 μg L^?1 day^?1 was obtained with 40 mg L^?1 selenate in the culture medium. Then a continuous cultivation process at several dilution rates was performed at 40 mg L^?1 selenate obtaining a maximum of 246 μg L^?1 day^?1 SeMet at a low dilution rate of 0.49 day^?1, calculated on total daily effluent volume. This paper describes for the first time an efficient long-term continuous cultivation of C. sorokiniana for the production of biomass enriched in the high value amino acid SeMet, at laboratory scale.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

Quantitative analysis of sesquiterpene lactones and thymol derivatives in extracts from Telekia speciosa

A high-performance liquid chromatographic method based on a gradient elution and the application of core–shell type stationary phase was developed to estimate contents of sesquiterpene lactones and monoterpenoid thymol derivatives in the tissues of Telekia speciosa. The detection and quantification limits of the analytes were 0.05–0.29 μg ml⁻¹ and 0.15–0.89 μg ml⁻¹, respectively. Calibration curves showed good linearities (R² > 0.9996) within the test ranges. Intra-day and inter-day precisions were satisfactory with RSD < 2.6%. The recoveries of the standards tested ranged from 96 to 107%. The overall time of analysis was less than 35 min. Using the method, seven major sesquiterpene lactones and one thymol derivative were quantified in different organs of the plant and plants of different origin. Aerial parts of T. speciosa accumulated miscellaneous sesquiterpene lactones, mainly of guaiane, pseudoguaiane, xanthane and eudesmane type, whereas roots of the plant contained almost exclusively isoalantolactone – an eudesmanolide of antiproliferative and anti-inflammatory activity (up to 1.2% dry weight). Flowers of T. speciosa proved to be an excellent source of xanthanolide – 8-epi-tomentosin (0.16–0.94%). Provenience of the plant material strongly influenced its biosynthetic potential.     

Enhanced accumulation of flavonolignans in Silybum marianum cultured roots by methyl jasmonate

Root cultures of Silybum marianum (L.) Gaertn. (Asteraceae) were established from in vitro germinated sterile plantlets. The cultures grew in hormone-free Murashige and Skoog medium. The flavonolignan content in the cultured roots was determined by HPLC using 30% acetonitrile in acidified water (0.5% phosphoric acid). The major flavonolignans produced by the cultured roots were silychristin (74.2 μg g^?1 fresh weight (FW)) and silydianin (8.1 μg g^?1 FW). The flavonolignan precursor taxifolin was also detected in the cultured roots (40.8 μg g^?1 FW). Addition of methyl jasmonate to 7-days-old root cultures for 48 h increased the content of the produced flavonolignans and taxifolin to approximately 300% of the control cultures. Methyl jasmonate also enhanced about sixfold the accumulation of a compound identified as 3,3′,5,5′,7-pentahydroxyflavanone.     

Serum selenium levels of the very low birth weight premature newborn infants with bronchopulmonary dysplasia

BackgroundThe selenium (Se) is an essential trace element that has a critical role in synthesis and activity of a number of selenoproteins with protective properties against free radical damage. This study was conducted to detect the serum Se concentration in very low birth weight (VLBW) preterm infants and its association with bronchopulmonary dysplasia (BPD).Materials and methodsCord blood Se concentration was determined in 54 neonates with gestation age 30 week or less. Another sample was obtained from these infants at day 28 of birth and serum Se levels were measured by atomic absorption spectrophotometer. All neonates were followed for oxygen dependency at 28 day after birth and 36 week postmenstrual age.ResultsThe mean cord blood Se concentration in studied neonates was 64.78 ± 20.73 μg L^?1. Serum Se concentration was 60.33 ± 26.62 μg L^?1 at age 28-day. No significant correlation was observed for serum Se concentration at birth and at one month after birth (r = ?0.04, p = 0.72). BPD was diagnosed in 25 neonates (46%). The mean serum Se concentration at one month was 57.16 ± 29.68 μg L^?1 in patients with BPD (25 cases) and 63.27 ± 23.6 μg L^?1 in 29 patients without BPD (p = 0.40).ConclusionIn our study, serum Se concentration at 28 day of birth was lower than cord blood levels in preterm neonates, but we have not found significant difference among patients who had BPD or not with respect to serum Se concentrations at this age.     

Morphology and bloom dynamics of Cochlodinium geminatum (Schütt) Schütt in the Pearl River Estuary,South China Sea

An unarmored dinoflagellate bloom of Cochlodinium geminatum (Schütt) Schütt has been identified in the Pearl River Estuary, South China Sea during the severe dry season, from late October to early November, 2009, when temperature and salinity ranged between 20.0–27.2 °C and 10.6–33.4, respectively. Light and scanning electron microscopy were used to identify the characteristics of C. geminatum and provided the clear morphological structure for this species. The organism was primarily found in chains of two cells or single cell, and no longer chains were observed. Cells were irregularly spherical or slightly dorso-ventrally, with size ranged between 28 and 36 μm and longer than wide. A large nucleus in the center with numerous golden chloroplasts was present, and the cingulum made 1.5 turns around the cell. The concentration of C. geminatum ranged from 10² to greater than 10⁷ cells l⁻¹ during the bloom period. Nutrient concentration ranges during the bloom were 1.29–81.00 μM NO₃, 0.14–12.14 μM NO₂, 0.21–6.29 μM NH₄, 0.23–6.26 μM PO₄ and 3.29–171.43 μM SiO₃, respectively. Total biomass expressed in terms of chlorophyll a ranged from 2.44 to 135.45 μg l⁻¹, with an average 19.9 μg l⁻¹ in surface water throughout the PRE. Two main clusters corresponding to the water sectors were defined with multivariate analysis (cluster and nMDS). Based on the composition and abundance of phytoplankton, spatial variations were observed at a significant level (ANOSIM, R = 0.44, P < 0.01). Although the pairwise correlation analysis detected no significant effect of any single environmental variable on the abundance of C. geminatum, the multivariate analysis (BIO-ENV) between biotic and abiotic variables resulted in the best variables combination with all measured factors involved (temperature, salinity, turbidity, NO₃, NO₂, NH₄, PO₄ and SiO₃) which showed a combined effect during the bloom of C. geminatum in the Pearl River Estuary (ρ_w = 0.477).     

Effect of arsenic on visible symptom and arsenic concentration in hydroponic Japanese mustard spinach

Responses of Japanese mustard spinach (JM-spinach; Brassica rapa L. var. pervirdis) were investigated at elevated levels of arsenic (As). Plants were grown hydroponically in the greenhouse under 0, 6.7, 33.5 and 67 μM As (equal to 0, 0.5, 2.5 and 5 mg L^?1 As, respectively) for 14 days. Arsenic was used as sodium meta-arsenite (NaAsO₂). Toxicity symptom was solely shown as shoot growth repression at 33.5 and 67 μM As exposures. Dry weight (DW) enhanced by 19.4% in shoot and 38.9% in root in the 6.7 μM As level as compared to control but decreased by 48.1% and 72.1% DW in shoot and 24.1% and 61.1% DW in root in the 33.5 and 67 μM As levels, respectively. This result indicated that As at lower concentration might have slight stimulating effect on JM-spinach growth, but toxicity increased with increasing As. Based on the regression lines between growth and As concentration in the plant tissues, the critical toxicity level (CTL) of As in JM-spinach shoot was 7.85 μg g^?1 DW considering 10% DW reduction. The CTL for the root was almost 2110 μg As g^?1 DW, indicating that shoot of JM-spinach was more sensitive to As-toxicity than that of root. Arsenic concentrations increased in plant parts with increasing As in the medium. Arsenic concentrations were also compared in DW and fresh weight (FW) basis. The JM-spinach concentrated unaccepted level of As in shoots for human consumption in the higher As levels without showing visible toxicity symptom. In spite of decreasing iron (Fe) concentration in shoot in the highest As level, chlorophyll index did not decrease accordingly. Phosphorus (P) concentration also decreased. Phosphorus concentration decreased much more than Fe concentration. Low P might help to mobilize Fe in shoots, resulting in higher chlorophyll index at 67 μM As level. Phosphorus might compete with Fe in shoot tissues of As-stressed JM-spinach.     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.