Metal binding compounds in hepatopancreas and haemolymph of porcellio scaber (isopoda) from contaminated and reference areas

1. In order to evaluate the role of metal-binding proteins in the tolerance mechanism of Porcellio scaber to heavy metals, a comparative study was made using isopods from three locations: a zinc-lead mine (Plombiéres), a zinc smelter (Budel) and a reference wood (Spanderswoud). The Cu, Zn and Cd concentrations and the protein composition were determined in the haemolymph and hepatopancreas from the isopods.2. A constant Cu/Zn molar ratio of about 5 was found in the haemolymph of all populations and no correlation was found between hepatopancreas and haemolymph Cu and Zn content.3. Using fast-performance liquid chromatography (FPLC), most of the haemolymph Cu and Zn appeared to be associated with a single UV absorbing peak corresponding with an apparent molecular weight of ± 70 kD; this peak is probably the monomer of hemocyanin.4. The hepatopancreas Zn and Cd concentration were elevated compared to the hepatopancreas of the smelter and mine isopods; after homogenization and centrifugation 70–80% of the metals were found in the supernatant.5. In all populations the hepatopancreas Cu-, Zn- and Cd-binding compounds eluted in separate peaks of low molecular weight, suggesting the absence of an MT-like compound in Porcellio scaber.6. The similarity of the protein profiles in haemolymph, and the similar distribution of the metals over the fractions in haemolymph and hepatopancreas suggests that inducible metal binding compounds are not involved in metal tolerance differences between populations.     

Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important r?le in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

Biosynthesis of an asparagine-linked oligosaccharide-containing calcitonin by a rat medullary thyroid carcinoma cell line

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.     

饥饿及再投喂对日本囊对虾糖代谢的影响

研究了日本囊对虾在饥饿和再投喂下血糖、肝胰脏糖原和肌糖原含量的变化.结果表明：在饥饿状态下,日本囊对虾肝胰脏糖原含量和血糖浓度在饥饿开始时迅速下降,肌糖原含量在饥饿10 d时下降到最低值,在饥饿10~15 d时通过糖原异生作用又恢复至最初水平,但随着饥饿时间的延长,糖原含量持续下降.恢复投喂后,肝胰脏糖原含量和肌糖原含量均能得到较好恢复,饥饿10 d和 15 d组的血糖浓度在恢复投喂10 d后显著高于对照组,但饥饿25 d组的血糖浓度始终显著低于对照.表明饥饿时间过长,对血糖浓度的恢复有较大影响     

Nature and distribution of copper in green lagoon crab Scylla serrata (Forskal)

In Scylla serrata (Forskal), copper is present in detectable quantities in all tissues except the central nervous system. 75% of animal's total copper is found in the haemolymph, hepatopancreas, and cuticle. Both the haemolymph and the hepatopancreas share equally ≈ 60% of the animal's total copper. Copper is found in the TCA-soluble and insoluble fractions of water-soluble tissue extracts and in the chloroform : methanol-soluble and insoluble fractions of water-insoluble residue from the hepatopancreas. No other tissue has all the fractions of copper. In haemolymph copper is present only in the TCA-insoluble fraction. The TCA-soluble fraction is also absent in muscles. The absence of any lipid-bound copper in other tissues and the presence of copper in all fractions from the hepatopancreas indicates that this tissue may play a major role in the mobilization, conservation and detoxication of copper.     

Quantitative variations during the intermoult cycle of peptides related to human growth hormone in Palaemon serratus (Crustacea; Decapoda)

Growth hormone-like peptides cross-reacting with an antiserum human specific were detected in the haemolymph, the midgut gland and stomach extracts of the prawn Palaemon serratus using radioimmunoassay. Parallelism was observed between the dilution sample curves and the hGH standard curve. Moreover, at least one peptide exhibiting a similar apparent molecular weight (22,000 daltons) with hGH was detected. In the three samples different amounts were detected during intermolt cycle: between 1.3 and 2.2 ng/ml in the haemolymph, 23 and 84 ng/g wet weight for the midgut gland, 12 and 71 ng/g ww for the stomach. The stages with highest values were respectively: D 1" and D 1"', D 1"' and D 2, D 3 and AB.     

Chronic effect of atrazine on hydromineral balance in the crab

Effect in vivo of atrazine on total body weight, hydration level, haemolymph volume, inorganic electrolytes was studied in the gill, hepatopancreas and haemolymph of the crab. Significant changes were seen in the tissues indicating disturbances in the hydromineral balance of the crab as a consequence of atrazine.     

Circadian variations in hemolymph lipids and lipoproteins and in hepatopancreatic lipids of the shrimp Penaeus japonicus

Lipids content of the haemolymph and the hepatopancreas in the decapod Crustacean P. japonicus exhibits a bicircadian rhythm characterized by one maximum in the night and another one during the day. The maximal values in the haemolymph are approximately two and a half times greater (8 mg/ml) than minimal ones (3 mg/ml). Variations are less important in the hepatopancreas. A bicircadian rhythm of lipid classes in the haemolymph is observed very significantly in concentration of polar lipids and free sterols with maximal values (6.87 mg/ml and 0.59 mg/ml) and minimal values (2.63 mg/ml and 0.23 mg/ml) respectively. Polar lipids are the major lipid fractions in the haemolymph (87%). The electrophoretic behaviour of haemolymph lipoproteins is determined.     

Variation in the free amino acid content of ovary, hepatopancreas and hemolymph of Penaeus schmitti during ovarian maturation (Crustacea, Decapoda, Penaeidae)

Quantitative and qualitative variations of free-amino-acids contents (FAA) of ovaries, hepatopancreas and haemolymph have been studied in the course of ovarian maturation in Penaeus schmitti. In the ovary, FAA content, expressed as microM/g ovary fresh weight, decreases from 56.2 to 23.6 microM between gonadosomatic index (GSI) 0.8 and GSI 7.1. There is an increase of hepatopancreas FAA content of 33.4% till GSI 4. Haemolymph FAA content increases from 0.32 to 1.76 microM/ml, with maximal values of 3.74 microM/ml when GSI are between 3 and 4. FAA content of ovaries, expressed as microM/g of animal, is 3.2 fold higher at the end of maturation, with a main increase in the first stages of ovogenesis. Maturation process has a quantitative effect on all FAA, but, on a qualitative point of view, it concerns mainly lysine, arginine and glycine. The main variations of AA composition (expressed as percentage of total FAA) are observed in haemolymph.     

Variations in protein levels in the hemolymph, hepatopancreas and ovary of Penaeus schmitti during ovarian maturation (Crustacea, Decapoda, Peneidae)

Variations of protein concentrations in the haemolymph, hepatopancreas and ovaries were studied during the ovarian maturation of the shrimp Penaeus schmitti. The main variations observed during this physiological process are the increase of protein and the decrease of water contents in the ovary, more pronounced at the first stages of gonadosomatic index (GSI:0-4). During ovarian maturation, water content of the ovaries and hepatopancreas decrease from 10.7 and 3.9% respectively. In relative values, protein content increases from about 40.0% in the ovary, 47.5% in the haemolymph and 15.4% in the hepatopancreas. In absolute values, the protein concentrations of the hepatopancreas showed no significant variation. In the opposite, ovary protein content is 14.7 fold higher at the end than the early stages of vitellogenesis. Relations between haemolymph, hepatopancreas and ovary with eventual transfers of protein material are discussed.     

Purification, isolation and search for possible functions of a phase-related 6080-Da peptide from the haemolymph of the desert locust, Schistocerca gregaria

Abstract. A novel 6-kDa peptide has been isolated from the haemolymph of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) , and fully identified. Its concentration is higher in crowd-reared (gregarious) animals than in isolated-reared (solitarious) ones. Its concentration decreases progressively from generation to generation with solitarization of gregarious locusts. The peptide is also present in freshly laid eggs. The concentration in eggs is higher in those from crowd-reared locusts. It is likely that the peptide is transferred from the female's haemolymph into the eggs because injection of the peptide into females before oviposition increases the amount of the 6-kDa peptide in the eggs. A two-step HPLC purification procedure for this peptide is described. It allowed several bioassays to test for a possible function. Although the peptide's concentration in the haemolymph is high (0.1 m m ), which suggests some physiological function, we have as yet not been able to identify a function. We hypothesize that the 6-kDa peptide may somehow play a role as a maternal factor in the determination of the phase-state of the offspring.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Isolation and characterization of a glycine-extended form of calcitonin receptor-stimulating peptide-1: another biologically active form of calcitonin receptor-stimulating peptide-1

In this study, we isolated a peptide eliciting a potent stimulatory effect on cAMP production in LLC-PK₁ cells from acid extracts of porcine brain. By structural analysis, this peptide was determined to be a C-terminal glycine-extended form of calcitonin receptor-stimulating peptide-1 (CRSP-1-Gly). Synthetic CRSP-1-Gly enhanced the cAMP production in COS-7 cells expressing calcitonin (CT) receptor as strongly as CRSP-1. Measurement of immunoreactive (IR) CRSP-1-Gly by radioimmunoassay using the specific antisera against CRSP-1-Gly showed that a relatively high level (>1 pmol/g wet weight) of IR-CRSP-1-Gly was detected in the midbrain, hypothalamus, anterior and posterior lobes of pituitary, and thyroid gland, and the ratio of IR-CRSP-1-Gly to total IR-CRSP-1 varies from 0.02 to 0.35 in each tissue. These results suggest that CRSP-1-Gly is actually present in the tissues as one of major endogenous molecular forms of CRSP-1, and can regulate the cells expressing the CT receptor both in the central nervous system and peripheral tissues in a manner similar to that of CRSP-1. IR-CRSP-2 and IR-CRSP-3 are also present in the brain and other tissues, but their tissue concentrations are 33% on average and less than 3% that of total IR-CRSP-1, respectively.     

Calcitonin-like immunoreactivity in the blue crab: tissue distribution, variations in the molt cycle, and partial characterization.

Calcitonin-like immunoreactivity has been found in blood and tissues of the marine blue crab, Callinectes sapidus Rathbun. The activity in blood rises significantly during the immediate premolt (D4) stage, but tissue immunoreactivity does not vary significantly with molt stage. The immunoreactivity to antisalmon calcitonin serum is due to a 27.2 kDa protein that shows strong similarities in amino acid composition to a similar protein in a lobster and to human calcitonin precursor. The protein is most abundant in the midgut glands (hepatopancreas), but is also found in significant quantities in several other tissues. Immunoreactivity in the blood appears to be primarily due to the same molecular weight fraction, with secondary contributions from smaller molecules, closer in size to vertebrate calcitonin. A physiological function of the calcitonin-like substance in calcium transport during or after molt has yet to be demonstrated.     

中华绒螯蟹对Pb和Cd的富集与释放特性

应用生物富集双箱动力学模型模拟了中华绒螯蟹(Eriocheir sinensis)分别在Pb浓度为0.25、0.50、0.75mg/L,Cd浓度为0.025、0.050、0.075mg/L的单一水环境中暴露时,蟹鳃、肝胰腺、肌肉和血淋巴对Pb和Cd的生物富集与释放实验,并通过非线性拟合得到中华绒螯蟹对Pb和Cd的富集速率常数k1、排出速率常数k2、生物富集系数BCF、生物半衰期B1/2、富集平衡时生物体内Pb和Cd含量CAmax等动力学参数。结果表明:(1)中华绒螯蟹对Pb和Cd具有明显的富集,蟹鳃、肝胰腺和肌肉中Pb和Cd的含量与富集时间和水环境中Pb和Cd暴露浓度表现出了很好的正相关,血淋巴在富集阶段没有明显的规律。理论平衡状态下鳃、肝胰腺和肌肉中Pb和Cd含量CAmax随着暴露浓度的增大而增大,且成正相关。(2)Pb和Cd在中华绒螯蟹组织器官中的富集具有选择性,开始实验前,Pb在中华绒螯蟹体内的的分布规律为:肝胰腺>鳃>肌肉>血淋巴;Cd的分布规律为:鳃>肝胰腺>血淋巴>肌肉。在实验浓度的Pb和Cd水环境中暴露16d后,Pb的分布规律为:鳃>肝胰腺>肌肉>血淋巴;Cd的分布规律为:肝胰腺>鳃>肌肉>血淋巴。(3)中华绒螯蟹对Pb和Cd的生物富集和释放都较缓慢。经过16d的生物富集,各组织器官中Pb和Cd的含量均未达到稳态平衡。Pb和Cd在组织器官中的生物富集系数(BCF)范围分别为5-51和6-3148,中华绒螯蟹对Cd的富集能力明显高于Pb(P1/2)范围分别为4-9d和8-57d,中华绒螯蟹对Cd的排出能力明显低于Pb。       

Calcium balance in premoult and post-moult Gammarus pulex (Amphipoda)

SUMMARY. Adult Gammarus pulex lose about 42% body calcium into solution over a 2–3-day period preceding the moult. A further 54% body calcium is lost with the exuviae, leaving c . 4% in the newly moulted animal.
Nearly all of the body calcium in a stage A (post moult) animal is contained in the hepatopancreas and a transient increase in the calcium content of this tissue is seen in some individuals. The haemolymph calcium compartment is heavily depleted at this stage. In stage B animals the hepatopancreas calcium level has returned to, or even fallen below, the intermoult level while the haemolymph calcium concentration remains lower than in intermoult animals.
Animals switch from a strongly negative calcium balance to a state of rapid calcium uptake immediately following moult. Uptake at 10°C proceeds at a rate of between 3 and 10 μmol g⁻¹ h⁻¹ depending on the calcium concentration of the external medium. The lower rate is found in starved animals in 0.1 mM calcium and this concentration is probably close to a minimum for the satisfactory restoration of body calcium. Recalcification is completed in 10–14 days in 0.1 mM calcium and is apparently enhanced by the presence of food in the form of a portion of oak leaf together with the cast exuviae. In 1.0 mM calcium the recalcification period is shortened to 3–4 days. This is considerably longer than the time recorded for French populations of this species (Vincent, 1969), although reasons for this are offered.     

嗜水气单胞菌感染对背角无齿蚌5种免疫相关酶活力的影响

本研究以背角无齿蚌为材料,利用嗜水气单胞菌为诱导刺激物,对背角无齿蚌进行注射感染,在注射后3、6、12 、24、48h分别取血淋巴、肝胰腺,测定其超氧化物歧化酶（SOD）、酚氧化酶 (PO)、酸性磷酸酶 (ACP)、碱性磷酸酶 (AKP) 及过氧化氢酶 (CAT) 的活力,并研究各项酶活力的变化规律。结果表明：除3h肝胰腺外,实验组的SOD活力均不同程度地高于对照组;血清实验组的PO活力开始显著高于对照组,然后降低,而肝胰腺实验组的PO活力持续高于对照组;另外,与对照组相比,实验组中血淋巴与肝胰腺的ACP、AKP和CAT活力在不同的时间段虽然有所增强,但两者之间无显著差异。因此,认为SOD、PO活性可以作为背角无齿蚌免疫抗病功能指标参数,而ACP、AKP及CAT活性能否作为该参数还有待于进一步研究。     

Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis--II. Subcellular distribution of metals and metal-binding proteins

Cd accumulation in the hepatopancreas and gills of the scallop resulted in an increase of metal concentration in the cytoplasm, while in control animals the general amount of Cd in the hepatopancreas was concentrated in the microsomes and cytoplasm. Cd appears to be equally distributed among high molecular weight proteins and metallothioneins in control animals. It was shown that during the experiment the metal was bound mainly to high molecular weight proteins. After 30 days of exposure of scallops to flowing water Cd was redistributed to metallothionein (MT)-like proteins. Cd concentration in the lipids of the hepatopancreas of control and experimental scallops was equal.