Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L^-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g^-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.     

Comparing Different Measures of Heat Resistance in Selected Lines of Drosophila melanogaster

Lines of the fly Drosophila melanogaster were selected for increased knockdown resistance to heat (39 degrees C) in a long tube. One set of lines was selected following prior heat hardening (1 h at 37 degrees C) and another without hardening. Each set consisted of three replicate selection lines and three unselected controls. Lines were tested for correlated responses to selection, in order to define the nature of knockdown resistance. Selection had a large effect on knockdown resistance, but selected lines did not differ from controls for knockdown time in small vials, survival, or recovery time following exposure to heat. Selection with and without hardening influenced the hardening response in the long tube, but not in small vial assays of resistance. The hardened selection lines had decreased resistance to ethanol and a reduced dry weight, whereas the non-hardened lines did not show these changes. The same correlated responses were also evident in two generation experiments on unselected flies. Both sets of lines showed a reduction in activity when tested at 37 degrees C, but not at 25 degrees C. These results indicate that different measures of heat resistance are surprisingly unrelated, and suggest that subtle features of the selective environment influence responses and correlated responses to selection. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5′-GUN-3′ tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.     

Foliar Spraying and Seed Soaking of Zinc Fertilizers Decreased Cadmium Accumulation in Cucumbers Grown in Cd-Contaminated Soils

It is important to use proper agronomic management to reduce cadmium (Cd) accumulation in plants, ensuring food safety. To find the most effective agronomic approach, the effect of foliar spraying and seed soaking of zinc (Zn) fertilizers on Cd accumulation in cucumbers (Cucumis sativus L.) grown in two soil Cd levels (2 and 5 mg kg^?1 Cd) with and without an immobilizing amendment (red mud, RM) was investigated in the present study. The results showed that the treatment of foliar Zn or seed Zn significantly decreased the Cd concentration in cucumber shoots by about 12–36% in Cd-contaminated soils without amendment. Combined with RM treatment, the foliar Zn treatment further decreased the Cd concentration in cucumber shoots by up to 48–66% in Cd-contaminated soils. There were significant negative correlations between Cd and Zn concentrations in shoots of cucumbers grown in soils treated with RM and foliar Zn. The results revealed that the cucumber seedlings treated with RM and foliar Zn had a higher capacity for limiting the transfer of Cd to aboveground tissues. The results also suggested that increasing seed Zn concentrations sufficiently might act as an efficient, economic, and practical method for decreasing Cd uptake in crops grown in mildly Cd-contaminated and Zn-deficient soils.     

镉诱导金属硫蛋白在华溪蟹组织中的表达

采用体外暴露染毒法,研究了不同浓度与时间条件下,镉诱导河南华溪蟹(Sinopotamon henanense)金属硫蛋白(metallothionein,MT)在肝胰腺、肌肉、鳃和卵巢中的表达差异。镉浓度分别为0、14·5mg/L、29mg/L和58mg/L;处理时间依次为1d、3d和5d。利用镉血红蛋白饱和法和火焰原子吸收分光光度法(AAS)测定MT的蛋白含量。结果显示,用不同的染毒浓度和处理时间,镉在组织中诱导产生MT的含量有较大差异,其中肝胰腺MT的诱导量最大,变化规律也最明显;肌肉中也有较大量MT的表达;而鳃和卵巢MT的诱导量均较低。此外,本文分析了镉的浓度与时间梯度对诱导MT表达的影响与毒性效应机制。结论:组织不同,染毒浓度及时间不同,镉诱导MT的表达也不同,具有一定的组织差异性和规律性。     

In Silico Analysis of Phosphoproteome Data Suggests a Rich-get-richer Process of Phosphosite Accumulation over Evolution

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers.Protein phosphorylation is an important and ubiquitous post-translational modification that regulates a variety of biological processes in various organisms (1–4). Reversible phosphorylations of serine, threonine, and tyrosine residues are critical steps in the control of signal transduction pathways (1–4). Recent advances in MS-based technologies and phosphopeptide enrichment methods have allowed high throughput and large scale in vivo phosphosite mapping for a wide variety of organisms such as human (5–8), mouse (9), yeast (10–12), fly (13,14), bacteria (15,16), and plants (17–19). Moreover information on several hundred to several thousand phosphosites from each study has been gathered in public databases such as Phospho.ELM (20), PhosphoSitePlus, PHOSIDA (21), and UniProt (22). However, the total number of phosphosites and most of their biological functions are still unknown. Similarly only about 10% of the estimated 500–600 human kinases target known phosphosite consensus sequences within their substrate proteins (23). Although the tyrosine phosphoproteome in Arabidopsis was recently published (24), the corresponding tyrosine kinases have not been identified because of the lack of known consensus sequences activated by tyrosine kinases.Computational data-mining approaches have been required to extract information from the large amount of accumulated phosphosite data obtained from experimental approaches. These approaches have also been used to add more meaningful information about each of the phosphosites to understand the proteome-wide protein phosphorylation in various organisms. One of the most useful strategies of computational data mining is to identify phosphorylated sequence motifs by extracting consensus sequences from the sets of amino acid sequences clustered around phosphorylated residues (25). A number of kinases and their corresponding recognition substrate motifs have been successfully identified by the experimental approach of incubating each target kinase with a combinatorial substrate peptide library and ATP, and these data are registered in various databases, including the Human Protein Reference Database (HPRD)¹ (26). With this knowledge of documented kinases and their related sequence motifs, we can use computational biology techniques to discover additional phosphorylated motifs in the numerous substrates shown in phosphoproteomics studies to be biologically phosphorylated. This has allowed us to reconstruct the kinome on a large scale (27–29).A comparative study of phosphoproteome data in multiple species has revealed that a wide range of phosphoproteins are relatively well conserved relative to non-phosphoproteins, and similarly many phosphoserine (Ser(P)), phosphothreonine (Thr(P)), and phosphotyrosine (Tyr(P)) phosphosites are well conserved compared with non-phosphorylated sites (21). Under natural selection, the emergence of phosphoproteins and the gain and loss of phosphosites should have changed the regulation of many intracellular systems, such as kinetic pathways, subcellular protein localization, and protein interactions and stabilization. This triggered our interest in the evolution of phosphoproteins and their phosphosites.In this study, we combined statistical physics and computational biology to investigate the role of selective pressure in the evolution of phosphoproteins and to create a model of the evolutionary gain of phosphosites. First using the human and mouse phosphoproteome data registered in public databases, we discovered that the number of phosphosites in each phosphoprotein follows a power-law distribution, which has been shown in complex systems science and statistical physics to emerge through a specific rich-get-richer process called preferential attachment growth (30–32). We therefore hypothesized that phosphoproteins may have evolved through a rich-get-richer process, gaining new phosphosites according to a probability density proportional to their current number of phosphosites. Starting from this hypothesis, we then explored how this particular evolutionary pattern may have arisen during natural selection and suggested that sets of phosphosites localized in limited spaces on protein surfaces may function as cooperative modules that are resistant to selective pressures. Therefore, to explain phosphosite evolution, we proposed a model in which the evolutionary gain of phosphosites follows a rich-get-richer process and evolution is promoted by the development of cooperative functional modules on protein surfaces.     

The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants through a Mechanism Independent of Mitophagy

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson''s disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.     

Metallothionein Gene Duplications and Metal Tolerance in Natural Populations of Drosophila melanogaster

A search for duplications of the Drosophila melanogaster metallothionein gene (Mtn) yielded numerous examples of this type of chromosomal rearrangement. These duplications are distributed widely--we found them in samples from four continents, and they are functional--larvae carrying Mtn duplications produce more Mtn RNA and tolerate increased cadmium and copper concentrations. Six different duplication types were characterized by restriction-enzyme analyses using probes from the Mtn region. The restriction maps show that in four cases the sequences, ranging in size between 2.2 and 6.0 kb, are arranged as direct, tandem repeats; in two other cases, this basic pattern is modified by the insertion of a putative transposable element into one of the repeated units. Duplications of the D. melanogaster metallothionein gene such as those that we found in natural populations may represent early stages in the evolution of a gene family.     

Eliminating Both Canonical and Short-Patch Mismatch Repair in Drosophila melanogaster Suggests a New Meiotic Recombination Model

In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.     

西番莲对镉、铅的吸收累积特性

以紫果西番莲和黄果西番莲为试材进行盆栽试验,研究镉、铅处理后西番莲体内镉、铅含量变化。结果表明,镉、铅在西番莲体内分布的一般规律是枝蔓> 叶> 果。不同品种西番莲的镉含量未见显著差异,而铅含量则呈显著差异。西番莲枝蔓和叶对镉、铅的吸收量分别随着各自处理浓度的增加而增加;统计显示,西番莲枝蔓和叶的镉、铅含量与各自土壤的添加量呈极显著正相关。值得注意的是,西番莲对镉有较强的吸收能力(枝蔓和叶对土壤镉的富集系数均大于1),而对铅的吸收能力较差。在本试验中,铅处理浓度为1 000 mg/kg时,西番莲果实可食部分铅的含量也低于无公害果品标准,说明西番莲作为果树生产时,即使在铅含量较高的污染土上种植,也是安全的;但要特别注意镉的污染风险。     

Comparative Genomics Suggests an Independent Origin of Cytoplasmic Incompatibility in Cardinium hertigii

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI–inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.     

Mechanisms of Cadmium Mobility and Accumulation in Indian Mustard

Indian mustard (Brassica juncea L.), a high biomass crop plant, accumulated substantial amounts of cadmium, with bioaccumulation coefficients (concentration of Cd in dry plant tissue/concentration in solution) of up to 1100 in shoots and 6700 in roots at nonphytotoxic concentrations of Cd (0.1 [mu]g/mL) in solution. This was associated with a rapid accumulation of phytochelatins in the root, where the majority of the Cd was coordinated with sulfur ligands, probably as a Cd-S4 complex, as demonstrated by x-ray absorption spectroscopy. In contrast, Cd moving in the xylem sap was coordinated predominantly with oxygen or nitrogen ligands. Cd concentrations in the xylem sap and the rate of Cd accumulation in the leaves displayed similar saturation kinetics, suggesting that the process of Cd transport from solution through the root and into the xylem is mediated by a saturable transport system(s). However, Cd translocation to the shoot appeared to be driven by transpiration, since ABA dramatically reduced Cd accumulation in leaves. Within leaves, Cd was preferentially accumulated in trichomes on the leaf surface, and this may be a possible detoxification mechanism.     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

脯氨酸对不结球白菜（Brassica chinensis）镉积累的影响

采用溶液培养的方法,研究了过量Cd对不结球白菜（Brassica chinensis）幼苗内源脯氨酸含量的影响及外源脯氨酸在不结球白菜Cd积累中的作用。100μmol？L-1 Cd处理明显增加不结球白菜叶片脯氨酸（Pro）的含量,并且随着Cd胁迫时间的延长,其含量也显著上升。100μmol？L-1 Cd处理明显降低脯氨酸脱氢酶（PDH）活性,而增加δ1-吡咯啉-5-羧酸合成酶（P5CS）的活性。1~5 mmol？L-1外源Pro处理显著降低不结球白菜幼苗地上部Cd含量,对根系Cd含量无显著影响,但外源Pro处理并不能缓解过量Cd对不结球白菜幼苗生物量的抑制作用。外源Pro处理下,不结球白菜叶片中Pro含量与其地上部干重呈显著的负相关关系。     

The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS. Special issue dedicated to Anthony Campagnoni.