Cadmium reduces zinc uptake but enhances its translocation in the cadmium-accumulator, <Emphasis Type="Italic">Carpobrotus rossii</Emphasis>, without affecting speciation

Background and aims

Interactions between Cd and Zn occur in soils and plants but are inconsistent. This study examined how Cd/Zn interactions influence the growth of Carpobrotus rossii (Haw.) and the accumulation of Cd and Zn in plants.

Methods

Plants were grown in nutrient solutions containing 5–100 μM Zn and 0, 5 or 15 μM Cd. Plant growth and tissue concentrations were measured, and the speciation of Zn within the plant tissues determined using synchrotron-based X-ray absorption spectroscopy.

Results

There was an additive negative interaction between Cd and Zn on root growth. Only the highest level of Zn (100 μM) decreased Cd concentrations in root and shoot tissues (by 40–64%), whilst 100 μM Zn enhanced Cd translocation at 5 μM Cd but decreased it at 15 μM Cd. In contrast, both 5 and 15 μM Cd decreased Zn concentrations in root and shoot tissues but increased Zn translocation by 30–90%. This interaction was not associated with changes in Zn speciation within the plants, with most Zn associated with oxalate (48–87%).

Conclusions

The presence of Zn and Cd resulted in an additive negative effect on root growth, but an antagonistic pattern in their accumulation in shoots of C. rossii.

     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

Rapid and efficient degradation of bisphenol A by chloroperoxidase from <Emphasis Type="Italic">Caldariomyces fumago</Emphasis>: product analysis and ecotoxicity evaluation of the degraded solution

Objectives

To degrade enzymatically bisphenol A (BPA) that causes serious environmental concerns and is difficult to be degraded by chemical or physical methods.

Results

BPA (150 mg l^?1) was completely degraded by chloroperoxidase (CPO)/H₂O₂ within 7 min at room temperature, atmospheric pressure with the enzyme at 6 μg CPO ml^?1. The degradation products were identified by HPLC–MS, which suggested involvement of multiple steps. Enzymatic treatment followed by existing bioremediation technologies (activated sludge) enhanced removal of COD from 9 to 54 %. Using an ecotoxicity evaluation with Chlorella pyrenoidosa, the degradation products had a lower toxicity than BPA.

Conclusion

BPA can be degraded rapidly and efficiently under mild conditions with chloroperoxidase at 6 μg ml^?1. The degradation products had a lower toxicity than BPA.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> endoglycosidase and sugar oxazoline

Objectives

To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.

Results

Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CC^N180H) were employed. pH 7.5 was ideal for both SG-oxazoline’s stability and Endo-CC’s transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 μg GlcNAc-RNase B, 200 μg SG-oxazoline and 3 μg Endo-CC^N180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min.

Conclusions

This transglycosylation method using SG-oxazoline and Endo-CC^N180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

     

Treatment of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> with 2-bromopalmitate alters morphology,endocytosis, differentiation and infectivity

Background

The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.

Results

The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC₅₀/48 h value for culture epimastigotes was estimated as 130 μM. The IC₅₀/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC₅₀/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.

Conclusion

Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.

     

Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Background

Insulin degrading enzyme (IDE) is a major protease of amyloid beta peptide (Aβ), a prominent toxic protein in Alzheimer’s disease (AD) pathogenesis. Previous studies suggested that statins promote IDE secretion; however, the underlying mechanism is unknown, as IDE has no signal sequence.

Results

In this study, we found that simvastatin (0.2 μM for 12 h) induced the degradation of extracellular Aβ₄₀, which depended on IDE secretion from primary astrocytes. In addition, simvastatin increased IDE secretion from astrocytes in a time- and dose-dependent manner. Moreover, simvastatin-mediated IDE secretion was mediated by an autophagy-based unconventional secretory pathway, and autophagic flux regulated simvastatin-mediated IDE secretion. Finally, simvastatin activated autophagy via the LKB1-AMPK-mTOR signaling pathway in astrocytes.

Conclusions

These results demonstrate a novel pathway for statin-mediated IDE secretion from astrocytes. Modulation of this pathway could provide a potential therapeutic target for treatment of Aβ pathology by enhancing extracellular clearance of Aβ.

     

Physiological and molecular analysis of aluminum tolerance in selected Kenyan maize lines

Aims

Aluminum (Al) toxicity is an important limitation to maize production in many tropical and sub-tropical acid soil areas. The aim of this study was to survey the variation in Al tolerance in a panel of maize lines adapted for Kenya and look for novel sources of Al tolerance.

Methods

112 Kenyan maize accessions were phenotyped for Al tolerance in solution culture. Several Al tolerance-related parameters including relative net root growth (RNRG), root apex Al accumulation, Al-activated root organic acid exudation, and expression of the maize Al tolerance gene, ZmMATE1, were used to classify Kenyan maize accessions.

Results

Based on RNRG, 42 %, 28 %, and 30 % of the lines were classified as highly tolerant, moderately tolerant and sensitive, respectively. Tolerant accessions accumulated less Al in their root apices compared to sensitive lines. The Kenyan maize line, CON 5, and the Brazilian standard for tolerance, Cateto, exhibited the greatest Al tolerance based on RNRG, but CON 5 had only about 50 % of ZmMATE1 gene expression relative to Cateto. CON 5 also had low root apex Al content and high citrate exudation, suggesting that it may employ a citrate transporter other than ZmMATE1.

Conclusions

We identified a very Al tolerant Kenyan maize line whose Al tolerance may be based in part on a novel tolerance gene. The maize lines identified in this study are useful germplasm for the development of varieties suitable for agriculture on acid soils in Kenya.

     

Molecular cloning and expression of a glycosyltransferase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> for modification of morin and related polyphenols

Objectives

To characterize glycosyltransferases from Bacillus subtilis ATCC 6633 and investigate their substrate specificity towards plant polyphenols.

Results

Among the cloned and expressed six UDP-glycosyltransferases (BsGT1-6), BsGT-1 showed activity with a wide range of polyphenols: morin, quercetin, alizarin, rehin, curcumin and aloe emodin. The gene of BsGT-1 has an ORF of 1206 bp encoding 402 amino acids. The recombinant enzyme was purified to homogeneity by Ni–NTA affinity chromatograph, and its biochemical characteristics were identified by HPLC–UV/MS, ¹H-NMR and ¹³C-NMR. BsGT-1 has an MW of approx. 46 kDa as indicated by SDS-PAGE; its activity was optimal at 40 °C and pH 8.5. The Km value of BsGT-1 towards morin was 110 μM.

Conclusions

BsGT-1 from B. subtilis was cloned. It had high catalytic capabilities towards polyphenols which would make it feasible for the structural modification of polyphenols.

     

The rapid accumulation of aluminum is ubiquitous in both the evergreen and deciduous leaves of Theaceae and Ternstroemiaceae plants over a wide pH range in acidic soils

Background and aims

Aluminum (Al) accumulator plants are occasionally found in certain genera or families of woody plant species that are broadly dispersed in the angiosperm phylogeny. However, spatial and seasonal patterns in Al accumulation within the closely related species of each group remain poorly understood.

Methods

We quantitatively monitored the internal Al levels of eight Theaceae and Ternstroemiaceae species growing on acidic soils at multiple sites.

Results

Among the eight species, seven other than Ternstroemia gymnanthera shared a rapid Al accumulation in the developing leaves. Species comparison revealed that Al accumulation in mature leaves saturates within a flushing year, regardless of differences in leaf structure, seasonality, and acidic soil pH (4.5–5.5) at multiple sites. In tall trees of Stewartia monadelpha, the Al contents of the leaves were constantly high irrespective of their height positions up to 12 m. Moreover, the Al content of the leaves was only slightly decreased in the last 2 weeks of autumn senescence, in which nitrogen (N) or phosphate (P) retranslocation had been completed.

Conclusion

These results suggest that most of the Theaceae and Ternstroemiaceae species possess an effective metal-transport mechanism that rapidly loads Al into the young leaves until each level reaches a species-specific threshold.

     

Effects of magnesium deficiency on magnesium uptake activity of rice root,evaluated using <Superscript>28</Superscript> Mg as a tracer

Aims

The mechanisms underlying magnesium (Mg) uptake by plant roots remain to be fully elucidated. In particular, there is little information about the effects of Mg deficiency on Mg uptake activity. A Mg uptake kinetic study is essential for better understanding the Mg uptake system.

Methods

We performed a Mg uptake tracer experiment in rice plants using ²⁸?Mg.

Results

Mg uptake was mediated by high- and low-affinity transport systems. The K _m value of the high-affinity transport system was approximately 70 μM under Mg-deficient conditions. The Mg uptake activity was promoted by Mg deficiency, which in turn fell to the basal level after 5- min of Mg resupply. The induced uptake rate was inhibited by ionophore treatment, suggesting that an energy-dependent uptake system is enhanced by Mg deficiency.

Conclusions

The Mg uptake changes rapidly with Mg conditions in rice, as revealed by a ²⁸?Mg tracer experiment. This technique is expected to be applicable for Mg uptake analyses, particularly in mutants or other lines.

     

A metabolomic,geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization

Background

Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective

This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

Methods

An NMR metabolomics approach was used to measure metabolite concentrations in 26 pollen extracts. Pollen count data was analyzed from five cities to model exposure.

Results

A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content: glucose, fructose, sucrose, and myo-inositol. In extracts of 10 mg of pollen/ml, the adenosine was highest for grasses (45 µM) followed by trees (23 µM) and weeds (19 µM). Pollen count data showed that tree pollen was typically 5–10 times the amount of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to be only 1.1, 0.11, and 0.12 µg, respectively. Seasonal models of pollen exposure and respiration suggest that it would be a rare event limited to tree pollen season for concentrations of pollen-derived adenosine to approach physiological levels.

Conclusion

Sugar content and other metabolites may be useful in classifying pollens. Unless other factors create localized exposures that are very different from these models, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.

     

The legacy of climate change effects: previous drought increases short-term litter decomposition rates in a temperate mixed grass- and shrubland

Aims

Fungi play a central role in litter decomposition, a key process controlling the terrestrial carbon cycle and nutrient availability for plants and microorganisms. Climate change and elevated CO₂ affect soil fungi, but the relative importance of the global change variables for litter decomposition is still uncertain. The main objective was therefore to assess the short-term litter decomposition and associated fungal community in a global change manipulated temperate heath ecosystem.

Methods

The heath had been exposed to 6 years of warming, elevated atmospheric CO₂ and an extended pre-summer drought. Litterbags with litter from heather (Calluna vulgaris) and wavy-hair grass (Deschampsia flexuosa) were incubated in the litter layer for 6 months, where after we analyzed the litter-associated fungal community, litter loss, CO₂ respiration, and total content of carbon, nitrogen and phosphorus.

Results

Elevated temperature tended to increase litter decomposition rates, whereas elevated CO₂ had no effect on the process. The pre-summer drought treatment had a positive impact on litter decomposition, CO₂ respiration and fungal abundance in the litterbags, although we observed no major changes in fungal community composition.

Conclusions

The drought treatment during pre-summer had a legacy effect on litter decomposition as decomposition rates were positively affected later in the year. The community structure of litter-decomposing fungi was not affected by the drought treatment. Hence, the legacy effect was not mediated by a change in the fungal community structure.