The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.     

Tratamiento y evolución del síndrome de tormenta de citoquinas asociados a infección por SARS-CoV-2 en pacientes octogenarios

IntroductionCytokine storm syndrome (CTS) is a serious complication of patients with SARS-CoV-2 infection. Treatment and evolution in octogenarians are not well defined. Our objective is to describe its clinical characteristics, the treatments and its clinical evolution.Patients and methodRetrospective observational study of consecutive patients admitted in the period between March 23 and April 12, 2020 with confirmed SARS-CoV-2 infection, with pneumonia by radiological study or chest tomography, whith STC criteria and who received treatment. We classified patients as those who received only glucocorticoid (GC) pulses, or GC and tocilizumab pulses. We determined serum levels of ferritin, CRP and D-dimers. The final variable was survival.Results21 patients, (80-88 years). The mean ferritin was 1056 microg/L (317-3,553), CRP 115.8 mg/dL (22-306) and D-dimers 2.9 m/L (0.45-17.5). All patients received GC pulses and in 2 cases simultaneously tocilizumab. The mean follow-up time was 13.7 days (8-21). The overall mortality was 38.1% (8/21 patients). The 2 patients who received tocilizumab died. The deceased had significantly higher levels of ferritin (1,254 vs. 925 microg/L; P = .045) and CRP (197.6 vs. 76 mg / dL; P = .007). At the end of the follow-up, a decrease in the biochemical parameters was observed with ferritin of 727 microg/L, CRP of 27 mg/dl and D-dimers of 1.18 mg/L. In 13/21 patients (61.9%), the CTS was controlled without the need to add other treatments.ConclusionsSTC mortality from SARS-CoV-2 is high despite treatment. A greater inflammatory response was associated with a higher mortality. Although it seems that the early use of GC pulses could control it, and the use of other treatments such as tocilizumab shouldo be, with the study design and its limitations, this conclusion cannot be stablished.     

High iron level in early pregnancy increased glucose intolerance

High iron stores in pregnancy are essential in preventing negative outcomes for both infants and mothers; however the risk of gestational diabetes mellitus (GDM) might also be increased. We intend to study the relationship between increased iron stores in early pregnancy and the risk of glucose intolerance and GDM. This prospective, observational, single-hospital study involved 104 non-anemic pregnant women, divided into 4 groups based on the quartile values for ferritin at the first trimester of pregnancy. All participants were screened for GDM with 75-g oral glucose tolerance test (OGTT) at 24–28 weeks’ gestation. We observed that ferritin levels at early pregnancy were significantly correlated to glucose level after OGTT at 1-h and 2-h (rho = 0.21, p < 0.05; rho = 0.43, p < 0.001 respectively). Furthermore, in the higher quartile for ferritin (>38.8 μg/L) glycemia at 2-h OGTT was significantly higher than in the others quartiles (p = 0.002). In multivariate regression analysis, serum ferritin was a significant determinant of glycemia at 2-h OGTT. Although we did not find a significant association in the incidence of GDM in women with higher serum ferritin levels, probably in reason to the limit power of our study, our data demonstrated that the role of iron excess is tightly involved in the pathogenesis of glucose intolerance. We report for the first time that high ferritin values in early pregnancy are predictors of impaired glucose tolerance in non-anemic women. Individual iron supplementation should be evaluated in order to minimize glucose impairment risk in women with high risk of diabetes.     

Increased whole blood manganese concentrations observed in children with iron deficiency anaemia

A prospective observational study was carried out at Alder Hey Children's Hospital, Liverpool, England, UK on children aged 1–6 years attending the pathology department for routine blood tests (n = 225). Whole blood manganese concentrations were measured plus the following markers of iron status; haemoglobin, MCV, MCH, RBC count, ferritin, transferrin saturation and soluble transferrin receptors. Multiple regression analysis was performed, with blood manganese as the dependent variable and factors of iron status, age and gender as independent variables. A strong relationship between blood manganese and iron deficiency was demonstrated (adjusted R² = 34.3%, p < 0.001) and the primary contributing factors to this relationship were haematological indices and soluble transferrin receptors. Subjects were categorised according to iron status using serum ferritin, transferrin saturation and haemoglobin indices. Children with iron deficiency anaemia had higher median blood manganese concentrations (16.4 μg/L, range 11.7–42.4, n = 20) than children with iron sufficiency (11 μg/L, range 5.9–20.9, n = 59, p < 0.001). This suggests that children with iron deficiency anaemia may be at risk from manganese toxicity (whole blood manganese >20 μg/L), and that this may lead to neurological problems. Treatment of iron deficiency in children is important both to improve iron status and to reduce the risk of manganese toxicity.     

Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynthesis pathway

Hyaluronic acid (HA) production in Streptococcus zooepidemicus competes for the carbon source along with biomass formation, lactate formation (via glycolysis) and pentose phosphate pathway (PPP). In our studies, increase in HA molecular weight was observed by redirecting the carbon flux towards HA biosynthesis pathway by partially inhibiting the glycolytic pathway. Batch bioreactor (1.2 L) studies showed that with the addition of 25 μM sodium iodoacetate, 5 g/L tryptophan and 10 g/L pyruvate, which are glycolytic inhibitors, HA molecular weight increased to 3.2, 3.2 and 3.1 MDa respectively compared to control run (2.4 MDa). Yield coefficients Y_HA/S and Y_LA/S showed inverse relationship, indicating competition for glucose between HA and lactic acid formation. Addition of 5 g/L glutamine along with 25 μM sodium iodoacetate also increased the HA concentration to 5.0 g/L from 2.0 g/L in control run. Metabolic flux analysis studies show that concentration and molecular weight of HA is increased by decreasing carbon flux towards glycolysis and PPP and increasing carbon flux towards HA precursor formation. It was observed that specific growth rate of the cells correlated positively to the specific HA production rate and negatively to the molecular weight of HA produced. Addition of antioxidant tannic acid also increased molecular weight to 3.0 MDa.     

Gender difference in blood cadmium concentration in the general population: Can it be explained by iron deficiency?

IntroductionGender differences in blood cadmium concentrations and the effect of iron deficiency on blood cadmium levels were analyzed in a representative sample of Koreans assessed in the Korean National Health and Nutritional Examination Survey (KNHANES) 2008–2011.MethodsA rolling sampling design was used to perform a complex, stratified, multistage probability cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. Serum ferritin was categorized as low (<15.0 μg/L), low normal (15.0–<30.0 μg/L for females and 15.0–<50.0 μg/L for males), and normal (≥30.0 μg/L for females and ≥50.0 μg/L for males), and its association with blood cadmium levels was assessed after adjustment for various demographic and lifestyle factors.ResultsThe geometric mean (GM) of the blood cadmium level was significantly higher in females than in males, and significantly higher in older individuals for both genders. After controlling for covariates, multiple regression analysis with interaction terms showed that blood cadmium was correlated with serum ferritin levels only in pre-menopausal females.DiscussionIron deficiency is associated with blood cadmium levels in a representative sample of pre-menopausal females, as evaluated in KNHANES. Gender differences in blood cadmium concentration may not be due solely to an iron deficiency-associated increase in blood cadmium.     

Chlorination-mediated EPS excretion shapes early-stage biofilm formation in drinking water systems

Microbial surface adhesion to surfaces and subsequent biofilm establishment are ubiquitous in drinking water systems, which often contribute to deteriorated water quality. Disinfectants are common agents applied to drinking water controlling microbial propagation, yet the underlying mechanisms of how disinfectants function to regulate microbial activity and thereby biofilm development remains elusive. We experimentally studied the effects of chlorination on extracellular polymeric substance (EPS) production, and its impacts on early-stage biofilm formation in a model drinking water system. Results showed that low-level chlorine (≤ 1.0 mg/L) stimulated microbial EPS (especially of proteins) excretion that favored early-stage biofilm formation. Microbes experiencing higher chlorination (>1.0 mg/L) exhibited clearly suppressed growth associated with reduced EPS release, consequently yielding less biofilm formation. Removal of cell-attached proteins and polysaccharides diminished biofilm formation, which highlighted the critical role of EPS (especially protein components) in biofilm development. A negative correlation between chlorination-mediated microbial protein production and cell surface charge suggested that chlorine disinfection may modify cell surface properties through regulation of microbial EPS excretion and thereby mediate biofilm formation. With these quantitative estimations, this study provides novel insights into how chlorination-mediated EPS excretion shapes early-stage biofilm formation, which is essential for practical functioning of drinking water systems.     

Aerobic degradation of 2-picolinic acid by a nitrobenzene-assimilating strain: Streptomyces sp. Z2

Streptomyces sp. Z2 was isolated from nitrobenzene contaminated activated sludge, which utilized nitrobenzene as a sole source of carbon, nitrogen, and energy under aerobic condition. It was found that besides nitrobenzene strain Z2 can degrade 2-picolinic acid. Strain Z2 completely degraded 2-picolinic acid with initial concentration of 500 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, and 3000 mg/L within 36 h, 50 h, 72 h, 100 h, 136 h, and 180 h, respectively. Kinetics of 2-picolinic acid degradation was described using the Andrews equation. The kinetic parameters were as follows: q_max = 3.81 h^?1, K_s = 83.10 mg/L, and K_i = 252.11 mg/L. During the biodegradation process, Z2 transformed 2-picolinic acid into a product which was identified as 6-hydroxy picolinic acid by UV–vis spectrometry, ¹H nuclear magnetic resonance spectroscopy, and mass spectrometry. 6-Hydroxy picolinic acid was then cleaved and mineralized with release of ammonia.     

The protective and anti-inflammatory effects of glucagon-like peptide-2 in an experimental rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease, that affects premature infants. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone and reduces the inflammation. We suspected that GLP-2 would have protective and anti-inflammatory effects in an experimental rat model of NEC. NEC was induced in newborn rats by enteral feeding with hyperosmolar formula, asphyxial stress and enteral administration of lipopolysaccharide (LPS). Rats were randomly divided into the following four groups: dam-fed, NEC, NEC + GLP-2(L) given 80 μg/kg/day of GLP-2, and NEC + GLP-2(H) given 800 μg/kg/day of GLP-2. GLP-2 was administered subcutaneously every 6 h before stress. All animals surviving beyond 96 h or any that developed signs of distress were euthanized. The clinical sickness score in the NEC + GLP-2(H) group was significantly lower than that in the NEC group. The NEC score and the survival rate in the NEC + GLP-2(H) group was significantly improved compared with those in the NEC and the NEC + GLP-2(L) groups. Villous height and crypt depth in both the GLP-2 treatment groups were significantly increased compared with those in the NEC group. There were no significant differences in the crypt cell proliferation indices among the groups. Ileal interstitial TNF-α and IL-6 level in the NEC + GLP-2(H) group was decreased to the same levels in the dam-fed group. High dose GLP-2 administration improved the incidence and survival rate for NEC. It also decreased mucosal inflammatory cytokine production. These results support a potential therapeutic role for GLP-2 in the treatment of NEC.     

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 ± 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 ± 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions — from 4 ± 1% to 7 ± 1% in the stable clone and from 10 ± 2% to 16 ± 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.     

Enhanced cephalotaxine production in Cephalotaxus mannii suspension cultures by combining glycometabolic regulation and elicitation

To study the combination effects of glycometabolic regulator NaF and elicitor methyl jasmonate (MJ) on cephalotaxine production in Cephalotaxus mannii suspension cultures, NaF of 10 mg/L, MJ of 100 μmol/L or both of them (NaF + MJ for short below) were added to the shake-flask cultures of C. mannii cell. It was found that NaF increased the activity of glucose 6-phosphate dehydrogenase (G6PDH), but had no significant effects on phenylalanine ammonium-lyase (PAL) activity and phenols formation. In contrast, MJ could activate PAL activity and led to phenols accumulation, but had no significant effects on G6PDH activity. To explore the effects of NaF and MJ on cephalotaxine biosynthesis, harringtonine and homoharringtonine, the two cephalotaxines, were analyzed in this work. The results obtained indicated that NaF + MJ treatment showed the strongest promotion of production in all tests. Harringtonine yield in NaF + MJ treated cells (7.245 mg/L) was 4.8-fold higher than that in control cells (1.506 mg/L), 1.7-fold that in NaF-treated cells (4.12 mg/L) and 1.6-fold that in MJ-treated cells (4.458 mg/L), respectively. No homoharringtonine was found besides in NaF + MJ treated cells (0.491 mg/L). With respect of the product release rates, they were 0%, 78%, 24% and 62% in control, NaF, MJ and NaF + MJ treatment, respectively. These results suggest that the combination of NaF and MJ had contributed to the synthesis and secretion of cephalotaxine in C. mannii cells.     

Effects of electromagnetic fields exposure on rapid micropropagation of beach plum (Prunus maritima)

In this research, electromagnetic fields of strength gradient 48–115 kA/m were applied, and it was found that the increase in the field strength stimulated the regeneration and growth in the explants. The effect of a 10-min treatment of explants with a strength of 97 kA/m on the regeneration and growth was highest, and the number of the sprouts induced (17.2 ± 1.74) was 2.3 times greater than that in the control group (7.40 ± 0.51). The fresh weight, dry weight and height of the sprouts from the explants treated with a strength of 97 kA/m, in culture medium 2 (MS + 2.0 mg/L ZT + 0.2 mg/L IBA + 30 mg/L Vc), were higher than those in culture medium 1 (MS + 2.0 mg/L ZT + 0.2 mg/L NAA + 30 mg/L Vc) and the control group, and the mean number of the sprouts in culture medium 2 increased 5.2 times, but 3.1 times in culture medium 1. Root length of plantlets from explants treated with a strength of 97 kA/m and optimized rooting medium 1/2MS + 0.1 mg/L NAA + 0.1 mg/L IBA + 30 mg/L bovine serum albumin (BSA) was significantly larger, compared with the control. The average root length was 5.39 ± 0.68 cm. It was concluded that the optimization of culture medium and treatment of explants with a certain magnetic field strength could increase the number of regeneration sprouts and growth.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

Optimization of a biotransformation process to produce 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin

The developed tandem biotransformation process for the directional biosynthesis of a designed compound 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin (4-TMP-DMEP) by Alternaria alternata S-f6 was systematically optimized. 28 °C of culture temperature and 120 rpm of rotary shaker speed were suitable for the accumulation of 4-TMP-DMEP. The production (i.e., 11.1 ± 1.4 mg/L) of 4-TMP-DMEP was remarkably improved by using an initial yeast extract concentration of 2.5 g/L. 2.0 g/L of Span 80 was beneficial for the 4-TMP-DMEP production (i.e., 25.0 ± 1.5 mg/L). Furthermore, the 4-TMP-DMEP production was remarkably improved by one pulse feeding of 50 mg/L of DMEP on day 6 and two pulse feedings of 40 mg/L of TMP on days 8 and 14 when its residual level was below 50 mg/L and 10 mg/L, respectively. The 4-TMP-DMEP production of 45.1 ± 1.6 mg/L was obtained in the fed-batch biotransformation process, which was enhanced by 726% and 256%, comparing to that (i.e., 5.4 ± 0.4 mg/L and 0.9 mg/L/day) obtained in the batch biotransformation before optimization.     

Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of Haemanthus L. (Amaryllidaceae)

The alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two alkaloids constituted 88, 91 and 98% of the total alkaloid extract from each species respectively. GC–MS analysis revealed that H. coccineus and H. sanguineus had a relative abundance of coccinine (74 and 91% respectively) to montanine (14 and 7% respectively); whereas H. montanus had 20% coccinine and 71% montanine. The three extracts and two isolated alkaloids were evaluated for binding to the serotonin transporter protein (SERT) in vitro. Affinity to SERT was highest in H. coccineus (IC₅₀ = 2.0 ± 1.1 μg/ml) followed by H. montanus (IC₅₀ = 6.8 ± 1.0 μg/ml) and H. sanguineus (IC₅₀ = 28.7 ± 1.1 μg/ml). Montanine (IC₅₀ = 121.3 ± 3.6 μM or 36.56 ± 1.14 μg/ml; K_i = 66.01 μM) was more active than coccinine (IC₅₀ = 196.3 ± 3.8 μM or 59.15 ± 1.08 μg/ml; K_i = 106.8 μM), both of which were less active than the total alkaloid extracts of each species investigated. The possible synergistic effects of two coccinine/montanine mixtures (80:20 and 20:80) were investigated, however the mixtures gave similar activities as the pure compounds and did not show any increase in activity or activity similar to the total alkaloid extracts. Thus the considerably higher activity observed in the total alkaloid extracts is not correlated to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line. This indicates that P-gp efflux will not be limiting for blood–brain-barrier passage of the alkaloids.     

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.     

Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicilium sizovae

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fructooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF₂15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C. cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC–PAD chromatography. C. cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), ¹F-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NMR analysis was identified as blastose [Fru-β(2 → 6)-Glc]. P. sizovae was very selective for the formation of ¹F-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.