Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida

Novel raw-starch-digesting and cold-adapted alpha-amylases (Amy I and Amy II) from the earthworm Eisenia foetida were purified to electrophoretically homogeneous states. The molecular weights of both purified enzymes were estimated to be 60,000 by SDS-PAGE. The enzymes were most active at pH 5.5 and 50 degrees C and stable at pH 7.0-9.0 and 50-60 degrees C. Both Amy I and II exhibited activities at 10 degrees C. The enzymes were inhibited by metal ions Cu(2+), Fe(2+), and Hg(2+), and hydrolyzed raw starch into glucose, maltose and maltotriose as end products.     

Accumulation and localization of lead in Eisenia foetida (Oligochaeta) tissues.

1. Accumulation and distribution of lead in tissues of Eisenia foetida (Oligochaeta) living in soils with different concentrations PbO2 was studied histochemically and with the use of the dithizione method. 2. The earthworms absorb lead from the soil through the alimentary canal as well as through the body wall. this leads to considerable accumulation of this metal in epithelial cells, especially in the epithelial gland cells of the body wall and of the alimentary tract. 3. High concentration of lead in the soil does not favour increased accumulation of lead in the earthworms' organisms. The histochemical data show that the most intense accumulation occurs when the concentration of PbO2 is 0-4 mg/1-00 g soil.     

赤子爱胜蚓（Eisenia foetida）中抗肿瘤与纤溶酶原激酶活性蛋白质的分离与鉴定

采用SephadexG 75和HiPrep 1 6 / 6 0DEAE离子交换等方法从赤子爱胜蚓 (Eiseniafoetida)中提取到一组活性蛋白质成分 ,双向电泳分析证明它们为一组pI在 3 .0～ 4 .0之间的酸性蛋白质 ;利用体外K5 6 2、HeLa、SY5Y等肿瘤细胞抑制实验和纤维蛋白平板实验 ,跟踪测定活性 ,证明乙醇沉淀组分D2 (8)是既具肿瘤抑制 ,又具有激酶活性的蛋白质成分。同时应用非变性电泳对乙醇沉淀组分进行分离 ,并利用电洗脱、凝胶原位酶解和ESI MS等蛋白质组学方法 ,鉴定了其中 6种蛋白质的分子量、氨基酸组成、N末端序列和肽质量指纹图信息 ,其中条带 9与D2 (8)组分为同一种蛋白质 ;研究证明蚯蚓中含有既具抗肿瘤活性又具有激酶活性的蛋白质成分。采用的方法可适用于活性蛋白质成分的整体分离与鉴定     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

赤子爱胜蚓对森林凋落物的分解效率

蚯蚓是帽儿山林区的主要土壤动物类群。饲养实验表明,赤子爱胜蚓(Eiseniafoetida)对不同种类、不同腐解程度的木本植物叶片的摄食量不同,对未分解叶的食性偏好依次为:白桦(Betulaplatyphylla)>杨(Populusdavidiana)>红松(Pinuskoraiensis)>蒙古栎(Quercusmongolica),对半分解叶的食性偏好依次为:杨>红松>蒙古栎>白桦,对同种叶片半分解叶的消耗能力大于未分解叶。这表明叶片种类和叶子腐化程度是影响蚯蚓摄食偏好的两个主要因素,混交林比纯林更有利于蚯蚓对难腐化叶片种类的分解。研究还表明,赤子爱胜蚓生物量在盛夏、春末以增重为主,夏初、秋初以失重为主,对落叶的消耗能力随种群生物量增大而增大,摄食量随温度的升高而增大,说明影响蚯蚓分解能力的另两个重要因素是种群生物量和温度。比较研究表明,从不适宜条件到适宜条件,蚯蚓消耗能力的理论范围约为6.378~27mg/(d·g)。能流研究表明,不同生境下,蚯蚓的各种生态效率变辐较大,即,毛增长效率:2.34%~9.92%,组织增长效率:28.95%~90.86%,同化效率:8.10%~34.27%,粪便率:65.73%~91.90%。赤子爱胜蚓的毛增长效率与同化效率约为该林区另一类主要土壤动物马陆的两倍,是帽儿山森林生态系统的重要分解者。因此,通过林木种类优化配置、温湿度条件的改变,可提高蚯蚓种群生物量及其分解能力,加快寒区厚积凋落物层的分解,加速林分更新。     

Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida

Earthworm fibrinolytic enzyme (EFE), a multi-com-ponent protease purified from some earthworm breeds,belongs to serine protease family with fibrinolytic activity[1]. It has been used in prevention and treatment of cardiacand cerebrovascular diseases in Ch…     

Iodination activity in Eisenia foetida (Annelida,Oligochaeta)

Summary In the nervous system of the earthworm Eisenia foetida, we have previously found thyroglobulin-like immunoreactive neurons. In the present study iodination activity was investigated by injecting worms or incubating them in vivo with radioiodine; animals treated with methimazole (MMI), an inhibitor of peroxidase-catalyzed iodination, served as controls. Radioiodinated proteins were identified in soluble extracts from ¹²⁵I-incubated animals; the sedimentation pattern of soluble proteins from cephalic segments showed a peak of radioactivity in the 3–4 S region. In animals pretreated with 10^-3 M MMI for 48 h, ¹²⁵I-incorporation into soluble proteins from cephalic segments was drastically reduced. Light-microscopic autoradiographic studies showed silver-grains selectively concentrated in the brain and ventral nerve cord of ¹²⁵I-injected animals. The highest grain-density occurred in the cerebral ganglion beginning 5 min after tracer injection; the reaction product was mainly distributed between the neurosecretory cells and neuropile fibres in the zone of the presumptive plexiform neurohaemal complex. In MMI-pretreated animals the reaction product was not visible in either cerebral ganglion or ventral nerve cord. Of interest, the setae appeared consistently positive with or without MMI treatment. These observations indicate that protein iodination involving peroxidase occurs in the nervous system of earthworms and suggest that iodination mechanisms, other than peroxidase-catalyzed, may be operating in some scleroprotein structures such as setae.     

Glycolipoprotein extract (G-90) from earthworm Eisenia foetida exerts some antioxidative activity

Antioxidants protect DNA, proteins and lipids in the body from damage. These types of damages are a major contributor to aging and to degenerative diseases such as cancer, cardiovascular disease, immune-system decline, brain dysfunction and cataracts. The effect of glycolipoprotein extract of Eisenia foetida (G-90) as an antioxidant was investigated in cultured human fibroblasts and epithelial cells. After treatment of the cells with H2O2 for 4 h, G-90 completely allows the cells to recover and stimulated their growth. When the cells were incubated with G-90 48 h before the treatment with H2O2, the oxidative damage of the cells did not occur. Thus, G-90 had an apparent protective effect against the toxicity of H2O2 and stimulated the growth of the cells. Ascorbic acid, a known antioxidant, did not allow the growth of the cells to recover after damage nor did it protect them, unless it was added simultaneously with H2O2. The antioxidative activity of G-90, together with its antibacterial and mitogen activities, could be useful in the study of G-90 as a wound-healing agent.     

Expression and characterization of earthworm Eisenia foetida lumbrokinase-3 in Pichia pastoris

Lumbrokinase-3 (LK-3, AY438622), first cloned from the earthworm Eisenia foetida in our laboratory, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the LK-3 gene was sub-cloned into yeast pPIC9K expression vector and transformed into the Pichia pastoris GS115 cells by electroporation. High level expression of LK-3 in yeast cells was confirmed with a different induction time. The activity of expressed LK-3 was observed in fibrin plates. In addition, the expressed LK-3 protein could dissolve fibrinogen and bovine serum albumin. The use of this system for the high level production of biological protein is implicated from this study.     

Nitrite biotransformation by mitochondria from the earthworm Eisenia foetida (Savigny).

1. Nitrite oxidase and cytochrome-c oxidase activity catalysed by cytochrome-aa3 were assayed in earthworms and rats. 2. Cytochrome-aa3 and intact mitochondria from the two species were anaerobically incubated in the presence of nitrite; the occurrence of mitochondria-induced nitrite biotransformations was evaluated by monitoring nitrite recovery in incubation medium. Possible nitric oxide production was also tested. 3. The ratio nitrite oxidase/cytochrome-c oxidase activity was much higher in earthworms than in rats. 4. Under anaerobic conditions and in the presence of respiratory substrates, earthworm mitochondria produced a time-dependent loss of nitrite in the incubation medium. On the contrary, rat mitochondria are unable to decrease environmental nitrite concentration. 5. Results support the notion that metabolic properties of earthworm mitochondria can be considered as an adaptation to chronic nitrite exposure, this toxicant being typically present in natural habitats of these worms.     

Zur Entstehung des Golgi-Apparates. Elektronenmikroskopische Untersuchungen an Spermatiden von Eisenia foetida (Annelidae)

Zusammenfassung An Spermatiden von Eisenia foetida wurde die Zelldifferenzierung zu Beginn der Spermiohistogenese untersucht. Die mit der zentralen, kernlosen Nährmasse, dem Cytophor, ein Syncytium bildenden Spermatiden besitzen zu Anfang der Zelldifferenzierung Vesikel, die im peripheren Kernbereich und im Lumen der perinucleären Zisterne liegen. Die Zellmembran am Verbindungsstück zum Cytophor trägt einen fuzzy coat, die Spermatidenmembran weist deutliche coated vesicles auf. Der Golgi-Apparat entsteht am prospektiven Schwanzende durch Auswachsen der ersten vesikelbildenden Lamelle aus der Kernmembran, die zuvor typische morphologische Veränderungen durchmacht. Die weiteren Lamellen eines Dictyosoms bilden sich durch Umfaltung der zuvor entstandenen. Anschließend werden an der Kernmembran die Mitochondrien differenziert. Die Genese der Cristae und der äußeren Mitochondrienmembran bleibt problematisch. Im Cytophor findet man annulierte Lamellen, degenerierte Golgi-Apparate und vesikelhaltige, abgeschnürte Zisternen des ER. Diese Formationen werden im Zusammenhang mit einer Reduktion des Cytoplasmas von den Spermatiden an das Cytophor abgegeben.

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On the origin of the golgi-apparatus. electron microscopical observations on spermatids of Eisenia foetida (Annelidae)

Summary The early cytodifferentiation is studied in developing spermatids of Eisenia foetida. The spermatids are attached to a central, anucleate mass of cytoplasm, the cytophore. They contain vesicles in the periphery of their nuclei and the perinuclear space. The cell membrane of the connecting bridge to the cytophore is covered with a fuzzy coat. The spermatid membrane is provided with coated vesicles. The Golgi apparatus develops at the prospective tailside by outgrowth of a vesicle producing lamella from the nuclear membrane, which before has undergone typical structural changes. Further dyctiosomal lamellae are formed by folding of already existing lamellae. Finally the mitochondria adhering to the nuclear membrane differentiate. The genesis of the cristae and the outer mitochondrial membrane remains problematical. In the cytophore there are annulate lamellae, degenerated dictyosomes and vesicle-containing cisternae of the endoplasmic reticulum. These formations are transferred from the spermatids to the cytophore, thus reducing the volume of their cytoplasm.

Herrn Prof. Dr. med. J. Staubesand danke ich für wertvolle Hinweise und Kritik.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

Le ghiandole calcifere (ghiandole di Morren) di Eisenia foetida. Studio istoe citochimico

Riassunto Sono riferiti alcuni reperti istochimici sulle ghiandole calcifere (ghiandole di Morren) di Eisenia foetida.Col metodo v. Kossa hanno assunto una colorazione nera più o meno intensa, oltre gli sferoliti e i romboedri di CaCO₃ elaborati dalle ghiandole, anche le assise secernenti.Il metodo Gomori per la fosfatasi alcalina rivela nelle lamine sinciziali delie ghiandole una attività fosfatasica di variabile intensità.La reazione PAS (Metodo McManus) e la reazione Gomori alla fucsina-paraldeide risultano positive nel citoplasma delle cellule esofagee, delle cellule delle cripte in continuità col lume esofageo che rappresentano la porzione anteriore delle ghiandole, e a livello delle fibrille interposte tra sincizio ghiandolare e lacune sanguigne. Specialmente la fucsina-paraldeide mette in rilievo anche la presenza di rare fibrille che attraversando le lacune sanguigne — il cui lume è di calibro molto variabile — ne connettono le pareti. Le stesse reazioni sono negative, tranne per qualche raro granulo, nelle assise secernenti.Il metodo Brachet per gli acidi nucleinici mette in evidenza una pironinofilia di vario grado nel citoplasma del sincizio, visibile sopratutto attorno ai nuclei. Anche i nucleoli, talvolta di dimensioni piuttosto cospicue relativamente alle dimensioni del nucleo, hanno pironinofilia più o meno accentuata, che scompare, come quella del citoplasma, dopo trattamento con ribonucleasi.La colorazione con blu di toluidina a p_H diversi o con tempi crescenti di colorazione permette di riconoscere delle granulazioni tenuemente ma nettamente metacromatiche anche a p_H più bassi o con trattamenti più brevi, mentre a livello del citoplasma in genere e nel nucleo, la colorazione metacromatica positiva si ha con le soluzioni a p_H relativamente più elevato o insorge gradualmente prolungando i tempi di colorazione. La metacromasia delle granulazioni è con tutta probabilità riferibile a mucopolisaccaridi acidi legati ai sali di Ca, mentre la colorazione diffusa del citoplasma è dovuta, verosimilmente, agli acidi nucleinici, la cui presenza è messa in rapporto col processo di continuo, cospicuo rinnovamento del sincizio ghiandolare durante il processo di secrezione.E' stato, infine, messo in evidenza, a conferma di osservazioni precedenti (Massal, 1929) la presenza nelle lamine secernenti di numerosissimi condriosomi filamentosi, disposti regolarmente, che conferiscono alle assise ghiandolari una struttura finemente striata.Questi reperti sono confrontati con quelli ottenuti con le stesse tecniche dall'A. sull'epitelio del sacco endolinfatico degli Anuri, anch'esso atto ad elaborare sali di Ca.Si sottolinea infine come ghiandole di Morren e sacco endolinfatico, organi appartenenti ad animali sistematicamente tanto lontani, hanno probabilmente, accanto ad alcune reazioni istochimiche comuni, anche funzioni in parte corrispondenti, in quanto in entrambi i casi l'accumulo di CaCO₃ potrebbe servire come riserva tampone per il mantenimento del p_H normale del sangue.     

Isolation and preliminary characterization of a Cd-binding protein from Tenebrio molitor (Coleoptera)

The effect of cadmium (Cd) exposure on Cd-binding ligands was investigated for the first time in a beetle (Coleoptera), using the mealworm Tenebrio molitor (L) as a model species. Exposure to Cd resulted in an approximate doubling of the Cd-binding capacity of the protein extracts from whole animals. Analysis showed that the increase was mainly explained by the induction of a Cd-binding protein of 7134.5 Da, with non-metallothionein characteristics. Amino acid analysis and de novo sequencing revealed that the protein has an unusually high content of the acidic amino acids aspartic and glutamic acid that may explain how this protein can bind Cd even without cysteine residues. Similarities in the amino acid composition suggest it to belong to a group of little studied proteins often referred to as "Cd-binding proteins without high cysteine content". This is the first report on isolation and peptide sequence determination of such a protein from a coleopteran.     

Isolation and characterization of a phospholipid transfer protein (LTP-II) from human plasma

In this report we have described the purification of a human plasma phospholipid transfer protein, designated LTP-II, which displayed the following characteristics: i) facilitated both the exchange and net mass transfer of lipoprotein phospholipids; ii) did not facilitate the transfer of lipoprotein cholesteryl esters (CE) or triglycerides (TG); iii) was not recognized by antibody to the human cholesteryl ester transfer protein (LTP-I); iv) showed no amino acid sequence homology to the cholesteryl ester transfer protein (LTP-I); v) has an apparent molecular weight (Mr) of 70,000 off Sephacryl S200, and 69,000 off sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); vi) has an apparent isoelectric point of 5.0 by chromatofocusing; and vii) when added to an incubation mixture of VLDL, HDL3, and the human plasma cholesteryl ester transfer protein (LTP-I), enhanced the observed transfer of cholesteryl esters from HDL3 to VLDL, even though LTP-II has no intrinsic cholesteryl ester transfer activity of its own. These results show that this phospholipid transfer protein is unique from the human plasma cholesteryl ester transfer protein, and may play an important role in human lipoprotein lipid metabolism.