Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.     

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.     

Cyperus rotundus suppresses AGE formation and protein oxidation in a model of fructose-mediated protein glycoxidation

Non-enzymatic glycation, as the chain reaction between reducing sugars and the free amino groups of proteins, has been shown to correlate with severity of diabetes and its complications. Cyperus rotundus (Cyperaceae) is used both as a food to promote health and as a drug to treat certain diseases. In this study, considering the antioxidative effects of C. rotundus, we examined whether C. rotundus also protects against protein oxidation and glycoxidation. The protein glycation inhibitory activity of hydroalcoholic extract of C. rotundus was evaluated in vitro using a model of fructose-mediated protein glycoxidation. The C. rotundus extract with glycation inhibitory activity also demonstrated antioxidant activity when a ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays as well as metal chelating activity were applied. Fructose (100 mM) increased fluorescence intensity of glycated bovine serum albumin (BSA) in terms of total AGEs during 14 days of exposure. Moreover, fructose caused more protein carbonyl (PCO) formation and also oxidized thiol groups more in glycated than in native BSA. The extract of C. rotundus at different concentrations (25–250 μg/ml) has significantly decreased the formation of AGEs in term of the fluorescence intensity of glycated BSA. Furthermore, we demonstrated the significant effect of C. rotundus extract on preventing oxidative protein damages including effect on PCO formation and thiol oxidation which are believed to form under the glycoxidation process. Our results highlight the protein glycation inhibitory and antioxidant activity of C. rotundus. These results might lead to the possibility of using the plant extract or its purified active components for targeting diabetic complications.     

A comparison of reproduction in co-occurring chthamalid barnacles, Chthamalus stellatus (Poli) and Chthamalus montagui Southward

Reproduction in Chthamalus montagui Southward and Chthamalus stellatus (Poli) has been compared along gradients of wave exposure and tidal height. The proportion of barnacles carrying egg masses was greatest at low shore levels in both species, although the difference between levels was more pronounced in C. stellatus. Both species were found to breed between the beginning of May and the end of September. The mean number of broods released per season in both species ranged from 1.0 to 2.1 at high shore levels, from 2.2 to 3.2 at mid shore levels, and from 2.6 to 4.4 at low shore levels. C. stellatus predominated low on the shore at an intermediately exposed site and on the mid and low shore at the most exposed site. At these places it produced more broods per year than C. montagui. Development of the embryos of the two species was complete after 3 wk in vivo at 15°C. Eggs containing embryos of C. stellatus were larger than those of C. montagui, and within each species, larger barnacles produced larger eggs. Egg masses of C. stellanus included more eggs than those of C. montagui. Brood size in both species was greatest in shelter and least in exposure. Egg production was, however, similar in the two species when adjusted for differences in body mass. Egg production per unit body mass was greatest at an intermediately exposed site.     

Long-term feeding effects of Catha edulis leaves on blood constituents in animals

In this study, the long-term (6 months) biochemical effects of varying levels of Catha edulis leaves on the plasma concentration of glucose, triglycerides, cholesterol, HDL-cholesterol, total protein, albumin, uric acid, urea and creatinine were examined. Our results demonstrated a significant decrease in plasma cholesterol throughout the treatment period by all levels of C. edulis leaves tested. This significant decrease in plasma cholesterol was halved at the end of the treatment period and corresponded with a significant increase in plasma HDL-cholesterol and a significant decrease in plasma glucose and triglycerides concentrations. Moreover, C. edulis treatment increased plasma uric acid significantly, in a time-dependent manner with the higher concentrations (20% and 30%) of C. edulis leaves. Only plasma albumin was decreased significantly at the end of the treatment period, with no significant effect on plasma total protein. This also coincided with a significant, dose-dependent decrease in plasma urea at month 6, with no significant effect on plasma creatinine concentration.     

Metallothionein-like proteins and zinc-copper interaction in the hindgut of Porcellio scaber (crustacea: Isopoda) exposed to zinc

Metallothioneins (MTs) are ubiquitous low-molecular-weight metalbinding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metaboliss of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 μg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of V _c/V_o≈2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber. are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.     

Differential susceptibility of subterranean termite castes to entomopathogenic nematodes

We examined the attachment and infectivity of two entomopathogenic nematode species, Steinernema carpocapsae and Heterorhabditis indica, on soldiers and workers in two subterranean termite species, Coptotermes formosanus and C. vastator. In attachment tests with S. carpocapsae, more nematodes attached to soldiers of C. formosanus and C. vastator in the absence of workers compared to soldiers that were in the presence of workers. In tests with soldiers alone, workers alone, and mixture of equal numbers of workers and soldiers, soldiers in the mixed groups had lower mortality than soldiers alone after 1 and 4 days for both termite species. Exposure of small groups of either termite species to S. carpocapsae resulted in higher mortality of soldiers after 1 and 4 days post exposure. Mortality in soldiers alone exposed to H. indica was not significant compared to mortality in mixed groups at 1 day exposure, but was significant after 4 days. In concentration-mortality tests, a significant two-way interaction existed between nematode concentration and termite caste for C. formosanus exposed to S. carpocapsae for 1 and 4 days. A significant effect of nematode concentration was found for C. vastator exposed to H. indica for 1 day. This termite species had lower mortality when exposed to H. indica after 1 and 4 days compared to C. formosanus. At 4 days post exposure to H. indica, the effects of nematode concentration and termite caste were significant. Steinernema carpocapsae caused higher mortality than H. indica, but mortality of workers was higher when exposed to H. indica. Soldiers of both species experienced rapid mortality when exposed to S. carpocapsae, whereas soldiers of C. vastator experienced lower mortality when exposed to H. indica. Thus, our results show that when soldiers alone or workers alone are exposed to the nematodes, there is a differential susceptibility of soldiers and workers to nematode infection with soldiers being more susceptible than workers. In a mixed group of soldiers and workers or workers alone, it appears that grooming behavior of the workers serves as a mechanism to reduce nematode infection of soldiers and workers. The reason for this differential response to nematode infection is that soldiers do not exhibit grooming behavior.     

Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas) leaves

Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and is classified into different types, which in general contains two domains (domain 1 and domain 2) with typical amino acid sequences (Rauser 1999). In this report two full-length cDNAs (Y459 and G14) encoding MT-like proteins were isolated from leaves of sweet potato (Ipomoea batatas). Their open reading frames contained 249 and 195 nucleotides (82 and 64 amino acids) for Y459 and G14, respectively, and exhibited a relatively low amino acid sequence similarity (ca. 25.8%). Gene structure studies showed that Y459 had the conserved domain 1 region of type 2 MT; however, the domain 2 region was not conserved and contained additional amino acids between the CxC and CxC spacing. G14 had conserved domains 1 and 2 of type 4 MT except that the last CxC of domain 2 was changed to RxC. Semi-quantitative RT-PCR showed that Y459 was expressed in significant quantity in roots and stems, but was much less in green leaves. During natural and induced (with dark and ethephon, an ethylene-releasing compound, treatments) leaf senescence, Y459 gene expression was significantly enhanced. In contrast, relatively constant gene expression levels were found for G14 in all tissues or treatments analyzed. In conclusion, the two MT-like protein genes of sweet potato display differential gene structures and gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

Natural interploidy hybridization among the key taxa involved in the origin of horticultural chrysanthemums

Understanding hybridization and introgression between natural plant populations can give important insights into the origins of cultivated species. Recent studies suggest differences in ploidy might not create such strong reproductive barriers as once thought, and thus studies into cultivated origins should examine all co-occurring taxa, including those with contrasting ploidy levels. Here, we characterized hybridization between Chrysanthemum indicum L., Chrysanthemum vestitum (Hemsley) Ling and Chrysanthemum vestitum var. latifolium (Zhou & Chen), the most important wild species involved in the origins of cultivated chrysanthemums. We analyzed the population structure of 317 Chrysanthemum accessions based on 13 microsatellite markers and sequenced chloroplast trnL-trnF for a subset of 103 Chrysanthemum accessions. We identified three distinct genetic clusters, corresponding to the three taxa. We detected 20 hybrids between species of different ploidy levels, of which 19 were between C. indicum (4x) and C. vestitum (6x) and one was between C. indicum and C. vestitum var. latifolium (6x). Fourteen hybrids between C. indicum and C. vestitum were from one of the five study sites. Chrysanthemum vestitum and C. vestitum var. latifolium share only one chloroplast haplotype. The substantially different number of hybrids between hybridizing species was likely due to different levels of reproductive isolation coupled with environmental selection against hybrids. In addition, human activities could play a role in the different patterns of hybridization among populations.     

Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress,dexamethasone, and metals

The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cuvieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids.     

亚热带米老排和杉木细根分解过程中养分与微生物群落组成的变化

对福建南平峡阳林场19年生米老排和杉木人工林的细根进行为期12个月的分解试验,研究不同树种分解过程中养分和微生物群落组成的动态变化,为理解亚热带不同人工林树种地下养分循环过程提供科学依据.结果表明: 米老排细根养分磷(P)、钾(K)初始含量显著高于杉木.分解过程中,两个树种细根P、K含量均显著降低,而细根氮(N)含量显著增加,且杉木细根N含量变化滞后于米老排.在分解过程中,杉木细根镁(Mg)含量无显著变化;米老排细根Mg含量变化显著,且在分解8个月时显著小于杉木.在分解过程中,真菌与细菌比值均显著表现为先升高后降低,且分解12个月时米老排细根真菌/细菌显著高于杉木.冗余分析表明,N(解释37.2%)、K(解释14.5%)含量和C/N(解释14.8%)是影响杉木细根分解过程中微生物群落组成变化的主要养分因子,而Mg(解释35.9%)和K(解释17.6%)含量则是米老排细根分解时影响微生物群落组成的主要养分因子.研究表明,在不同树种中,除了N之外,Mg等其他养分元素也可能是影响根系分解的重要因子.     

Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.     

Caffeine metabolism in Coffea arabica and other species of coffee

High performance liquid chromatography has been used to measure the quantities of caffeine, theobromine, and theophylline in aqueous extracts of endosperm from immature and mature fruits of Coffea arabica and six other species of Coffea. Caffeine was the alkaloid present in largest amounts and, with one exception, in concentrations that were broadly similar in immature and mature fruit. The highest concentrations of caffeine were found in C. canephora at 35.1 and 24.5 mg g⁻¹, respectively, in immature and mature endosperm. The lowest concentrations were in C. bengalensis, where caffeine was not detected in extracts from mature fruit. [8-³H]Caffeine was metabolised relatively slowly by immature endosperm of C. arabica and C. canephora. In contrast, C. dewevrei, C. eugenioides, C. stenophylla, C. salvatrix and C. bengalensis all appeared to metabolise [8-³H]caffeine much more rapidly, as the percentage recovery of the applied label was much lower and there was more extensive incorporation of radioactivity into theobromine, theophylline, 3-methylxanthine and two unidentified polar metabolites. The endogenous caffeine concentrations and the metabolism data indicate that there may be marked differences in the rate of turnover of caffeine in the various species of Coffea. Potential sources of material for the production of naturally decaffeinated coffee are discussed.     

Metallothionein induction and heavy metal accumulation in white shrimp Litopenaeus vannamei exposed to cadmium and zinc

Metallothioneins (MTs) have been widely considered for their potential use as specific biomarkers to reflect the existence of heavy metal pollution, because their induction has been observed to be obviously elevated after heavy metal exposure in a large number organism studied. However, relatively fewer efforts have been made in MT-related studies of prawn species, such as the white shrimp Litopenaeus vannamei, a globally important aquaculture species. With the results from gel filtration chromatography, we demonstrate the existence of MTs or MT-like proteins in L.vannamei. We further studied the relationship between MT induction and metals accumulation after long-term exposure to the heavy metals Cd and Zn. From our results, it is very clear that the response of L. vannamei to Cd differs from that to Zn, and this should be considered when using MTs in field applications to monitor metals contamination.     

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl^-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.     

A human metallothionein pseudogene containing AG/CT repetitive elements

Summary A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5 regulatory region, the 5 untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10–31 encoding a portion of the -domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32–61, representing the complete -domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.     

Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands

The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type in The Netherlands.