Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Changes in isotypes and enzyme activity of apyrase during germination of dark-grown pea (Pisum sativum) seedlings

In the present study we used 2D-PAGE and Western blotting to investigate the expression of different isotypes of apyrase (EC 3.6.1.5) during imbibition, germination and initial growth of pea ( Pisum sativum L . var. Alaska) seedlings in the dark. The 49 kDa apyrase was absent in the 10-h imbibed embryos, but began to appear after 16 h germination and increased with germination time. By 62 h, there were five isotypes present at pI 5.8, 6.0, 6.3, 6.6 and 6.8, with those at pI 6.0, 6.3, and 6.6 being most abundant and the one at pI 6.3 predominating, whereas the most acidic and basic isotypes were only present in significant amounts in seedlings after 62 h germination. Stems contained all five isotypes and had more pI 6.0, 6.3 and 6.6 isotype than the plumules, whereas in the roots there were very small amounts of all isotypes. Partial amino acid sequencing showed that all isotypes were identical with apy1, not the more recently described apy2. Apyrase activity was absent in imbibed embryos, but increased sharply during germination and reached a maximum after 62 h. Based upon the capability of the enzyme to hydrolyse ATP, CTP, GTP, TTP, UTP, and ADP (but not AMP), its susceptibility to various ATPase inhibitors, and coincidence of expression of the protein and enzyme activity, we estimate that 50–70% of the ATPase activity results from the 49 kDa apyrase. The present results suggest that isotypes of pI 6.0, 6.3, and 6.6 are physiologically important and strongly indicate a crucial role for apyrase in the differentiation and development of pea seedlings.     

Sub-cellular distribution and isotypes of a 49-kDa apyrase from Pisum sativum

We isolated a 49-kDa protein from various sub-cellular fractions from pea (Pisum sativum L. var. Alaska) stems using heparin affinity and cation exchange column chromatography. The corresponding proteins from all these fractions were identified as apyrase (EC 3.6.1.5) because they hydrolyzed both nucleoside tri- and diphosphates into their respective monophosphates. Using an antibody raised against apyrase, we studied the enzyme’s sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50%), the supernatant (33%), the cytoskeleton (14%), and the nuclei (3%). Immuno-electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures in the cytoplasm associated with ribosomes. Even though there is only one gene (with two alleles), for this protein, 2D gels indicated there were at least five isotypes, three being major, and the relative abundance of these isotypes differed in different fractions. Enzymes from all fractions: (a) hydrolyzed nucleoside triphosphates and diphosphates, but not monophosphates, (b) were insensitive to most ATPase inhibitors (azide, fluoride, nitrate, molybdate, ouabain, quercetin), but (c) were all inhibited by vanadium pentoxide at relatively high concentrations. There were, however, some subtle differences between enzymes from different sub-cellular fractions, including different ADP/ATP hydrolysis ratios. These results show that the 49-kDa apyrase is located in various compartments within the cell (cell wall, nuclei, and the cytoskeleton) and that the enzymes from all fractions are basically similar in their apyrase function. We suggest that the enzyme is modified in various ways to furnish different forms with different (non-apyrase) functions in different sub-cellular locations.     

7500 yr of mire-pool development and the history of Pinus sylvestris (L.) in Sub-Arctic coastal Norway

In coastal North Norway, mire areas and mire pools frequently exist, but their development and time of origin are poorly known. In order to investigate the development of a coastal mire pool and relate its changes to known climatic changes, a sediment sequence from the mire-pool Lillevardhaugvatnet (c. 0.05 ha large), was ¹⁴C-dated, investigated for loss on ignition and analysed for pollen and botanical macrofossils.The bottom of the sequence dates c. 7500 cal. BP. The site gradually developed from a swamp forest c. 6200 cal. BP via a more open dwarf-shrub phase to a pool c. 5000 cal. BP. The pool grew in depth as the result of continuous peat growth damming the water body. The water level of the pool was probably lowered by erosional drain c. 2700 cal. BP. Redeposited peat in the sequence occurred c. 2100 cal. BP and c. 1100 cal. BP. ¹⁴C-dates and pollen indicate that erosion and redeposition of the peat surrounding the mire pool is a normal process, connected with the expansion of the water body.The combination of macrofossils and pollen accumulation rates (PAR) of P. sylvestris indicates that in small lakes in coastal areas of North Norway, a PAR of 200–400 cm^− 2 yr^− 1 is sufficient for indicating local presence of P. sylvestris. P. sylvestris is represented with abundant macrofossils between 4800 and 2100 cal. BP. It is suggested that a marked P. sylvestris decrease about 2100 cal. BP may be a combined effect of human impact and climatic deterioration. A possible final termination of the P. sylvestris population about 1600 cal. BP may be considered human-made.     

Production of recombinant protein and polyclonal mouse antiserum for ferritin from Sipuncula Phascolosoma esculenta

The iron storage protein, ferritin, plays a key role in iron metabolism, but its regulation and functions in many invertebrate species are still largely unknown. In our previous work, an inducible ferritin cDNA from Phascolosoma esculenta with a full-length of 1017 bp has been cloned. In this follow-up study, the deducted ferritin protein sequence was predicted to be a polypeptide of 175 amino acids with a molecular mass of 20.1955 kDa and an isoelectric point of 5.08. The cDNA sequence of P. esculenta ferritin was constructed into pET system expression system and efficiently expressed in E. coli BL21 under IPTG induction. The recombinant ferritin was detected as a 24 kDa protein by SDS-PAGE. After purification directly from the gel, the recombinant ferritin was used to immunize mice and the anti-serum was prepared. The antibody displayed a strong immunological reactivity and specificity when used in Western-blot analysis. For the first time, our work provided a set of molecular tools essential for the further studies of ferritin protein functions in P. esculenta.     

Regulation of the utilization of 4-hydroxybenzoate and 4-hydroxycinnamate in batch and continuous cultures of Pseudomonas testosteroni

Pseudomonas testosteroni metabolized 4-hydroxycinnamate by an initial cleavage of the side chain to yield acetate and the aromatic moiety, 4-hydroxybenzaldehyde. The latter was further oxidized via 4-hydroxybenzoate to protocatechuate, which underwent meta cleavage. During growth of the organism on 4-hydroxycinnamate, the for acetate showed an undulating pattern, which was attributed to alternating induction and repression of enzymes involved in the oxidation of acetate. Repression was caused either by 4-hydroxybenzoate or by its later metabolites, formate and pyruvate.In batch culture, P. testosteroni oxidized mixtures of 4-hydroxybenzoate and 4-hydroxycinnamate in a diauxic pattern. The capacity to oxidize 4-hydroxycinnamate appeared in the cells before 4-hydroxybenzoate was exhausted, indicating that the enzymes catalysing the conversion of 4-hydroxycinnamate into 4-hydroxybenzoate. were induced despite the presence of 4-hydroxybenzoate. The induction of these early enzymes of 4-hydroxycinnamate catabolism started when the molar concentration ratio of 4-hydroxybenzoate to 4-hydroxycinnamate fell below a value of 0.3.In continuous culture of P. testosteroni on a mixture of 4-hydroxybenzoate and 4-hydroxycinnamate, both substrates were almost completely utilized up to a dilution rate of about 0.5/h. At higher dilution rates, 4-hydroxycinnamate was decreasingly utilized so that eventually at a dilution rate of 0.74/h, its effluent concentration equalled its influent concentration. At D _M, a utilization ratio of 1.23 in favour of 4-hydroxybenzoate was found to become established in the culture. The of the cells for acetate was maximal at a dilution rate of 0.38/h and decreased before 4-hydroxycinnamate utilization was at its peak at 0.59/h. This suggested that it was mainly the aromatic moiety of 4-hydroxycinnamate which was metabolized at high dilution rates. The failure to utilize acetate at high dilution rates was apparently due to the repression of its catabolic enzymes by later metabolites of 4-hydroxybenzoate and to the relatively low concentration of acetate in the fermenter. This low concentration, due to the continuous washout of acetate, prevented it from relieving the repression.Abbreviations 4HB 4-hydroxybenzoate - 4HC 4-hydroxycinnamate - D _M dilution rate allowing maximal cell output rate - OD optical density     

Identification of low molecular weight molecules as new components of the nacre organic matrix

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da–700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.     

Testosterone metabolism in Neomysis integer following exposure to benzo(a)pyrene

Cytochromes P450 (CYPs) are important enzymes involved in the regulation of hormone synthesis and in the detoxification and/or activation of xenobiotics. CYPs are found in virtually all organisms, from archae, and eubacteria to eukaryota. A number of endocrine disruptors are suspected of exerting their effects through disruption of normal CYP function. Consequently, alterations in steroid hormone metabolism through changes in CYP could provide an important tool to evaluate potential effects of endocrine disruptors. The aim of this study was to investigate the potential effects of the known CYP modulator, benzo(a)pyrene (B(a)P), on the testosterone metabolism in the invertebrate Neomysis integer (Crustacea; Mysidacea). N. integer were exposed for 96 h to 0.43, 2.39, 28.83, 339.00 and 1682.86 μg B(a)P L^− 1 and a solvent control, and subsequently their ability to metabolize testosterone was assessed. Identification and quantification of the produced phase I and phase II testosterone metabolites was performed using liquid chromatography coupled with multiple mass spectrometry (LC–MS²). Significant changes were observed in the overall ability of N. integer to metabolize testosterone when exposed to 2.39, 28.83, 339.00 and 1682.86 μg B(a)P L^− 1 as compared to the control animals.     

Preservation of Giardia cysts in stool samples for subsequent PCR analysis

Genotyping of Giardia duodenalis cysts in faecal samples has become a regularly employed tool by researchers investigating different aspects of the epidemiology and pathology of Giardia infection in human and animal populations. However, such investigations are often limited to some extent by lack of PCR amplification from a proportion of the samples, and this often seems to be associated with the storage medium used for the samples. Various different storage media have been used in different studies, but investigation of which storage media are most appropriate and which may compromise subsequent PCR investigations has not been systematically explored to date.In this study, 4 different, commonly used storage media were investigated for their effects over time on subsequent PCR amplification of DNA from Giardia cysts in stool samples. Microscopic examination of the samples and real-time PCR were used to investigate 7 different samples over a period of 3 months. Our findings indicate that storage in ethanol or potassium dichromate at 4 °C gave the best results and, that if immunomagnetic separation was used prior to PCR (as may be appropriate for samples with low cyst numbers), then storage in potassium dichromate gave the best results.     

Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 μg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 ± 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T₁ were fed on basal diet (control). The fish of T₂ were given the basal diet, containing propolis-ethanolic-extract. The fish of T₃ were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 × 10⁷ cells ml⁻¹) at the end of the feeding period and kept for 15 more days.The best growth rate and feed conversion ratio were obtained with T_2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T₂ followed by T₃ when compared with the control group. The HCT-level and monocyte-counts were increased (T₂). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T₂ and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T₂. The RLP against A. hydrophila was high with T₂ and T₃.The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.     

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 °C to 0 °C, 8 °C, 16 °C, 24 °C, and 32 °C for 72 h) and salinity changes (from 30‰ to 20‰, 25‰, and 35‰ for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25‰ and 35‰) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 °C. At 32 °C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 °C for 0.5 h exposure, and increased significantly at 32 °C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 °C in 0.5 h to 6 h exposures and at 8 °C for 1 h. By contrast, activities of MPO decreased significantly in 24 °C and 32 °C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 °C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20‰. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses.     

The regulation of K+ influx in excised barley roots

The electrochemical potential differences for potassium, between excised barley (Hordeum vulgare L.) roots and external media containing 0.05 mM KCl+0.5 mM CaSO₄, were determined over a 4-h period during which initially low-K⁺ roots accumulated K⁺ by pretreatment in 50 mM KCl plus 0.5 mM CaCl₂. This pretreatment resulted in increased internal [K⁺], decreased K⁺ influx (as measured from 0.05 mM KCl+0.5 mM CaSO₄) and decreased values of . These observations indicate that the decline of K⁺ influx associated with increased internal K⁺ concentration cannot be accounted for by passive adjustment to the electrochemical gradient for this ion.     

Characterization of natural rubber biosynthesisin Ficus benghalensis

Natural rubber was identified for the first time in the latex of Ficus benghalensis, and the rubber biosynthetic activity in latex and rubber particles was investigated. ¹³C NMR analysis of samples prepared by successive extractions with acetone and benzene confirmed that the benzene-soluble residues were natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of F. benghalensis was approximately 17 %. Gel permeation chromatography revealed that the molecular mass of the natural rubber from F. benghalensis was approximately 1 500 kDa. The high rubber content and large molecular size suggest that F. benghalensis is a good candidate for an alternative rubber source. Examination of latex serum from F. benghalensis by SDS-polyacrylamide gel electrophoresis revealed a small number of proteins with major proteins of 31 and 55 kDa in size. The 31-kDa protein was predominant in catalytically-active rubber particles. Determination of metal ion concentration in latex and a comparison of the effect of ethylenediamine-tetraacetic acid on in vitro rubber biosynthesis in F. benghalensis, F. carica and Hevea brasiliensis suggest that the divalent metal ion present in latex serum is an important physiological factor controlling the rubber biosynthetic activities in these plant species. Microscopic examination revealed that the rubber in F. benghalensis occurred in a series of laticifer cells located in concentric zones in the inner bark of stems and branches.     

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis

We investigated the trans-lactational maternal–neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 μg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.     

Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K_cat = 3.2 pmol/min/pmol P450, K_m = 36 μM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.     

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.     

Molecular cloning and characterization of a glycosyl hydrolase family 9 cellulase distributed throughout the digestive tract of the cricket Teleogryllus emma

A novel endogenous β-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 °C. The K_m and V_max values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract.