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First published online February 20, 2004
Journal of Experimental Biology 207, 1101-1111 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00858

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Stimulation by cadmium of myohemerythrin-like cells in the gut of the annelid Nereis diversicolor

Sylvain Demuynck^1,*, Beatrice Bocquet-Muchembled², Laurence Deloffre², Fabien Grumiaux¹ and Alain Leprêtre¹

¹ Laboratoire d'Ecologie Numérique et d'Ecotoxicologie UPRES EA 3570, FR 1818 CNRS
² Laboratoire de Neuroimmunité des Annélides UMR 97, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cedex, France

View larger version (14K): [in a new window]   Fig. 1. Gel-permeation chromatography of acute Cd-exposed (20 mg l–1, 2 days) N. diversicolor gut supernatant. Column: Hi- Load Superdex 75 Prep grade (Pharmacia); sample volume: 2 ml; flow rate: 1 ml min–1; elution buffer: 10 mmol l–1 Tris-HCl, pH 8.6. (A) The absorbance profile at 280 nm shows three main protein peaks, I, II and III. (B) Analysis of the Cd content (µg ml–1) of the chromatographic fractions shows that this metal is associated with components eluted in the void volume of the column (>70 kDa) likely to represent the hemoglobin (peak I in A) and components of the second main peak (peak II in A). (C) Enzyme-linked immunosorbent assay (ELISA) using the mAb anti-MPII revealed that peak II fractions contain the Cd-binding protein MPII.

View larger version (26K): [in a new window]   Fig. 2. SDS-polyacrylamide gradient gel electrophoresis (SDS PAGGE) (5–25% acrylamide gradient slab gel) under reducing conditions. (A) Gel stained with Coomassie Brilliant Blue R250 (lanes 1 and 2) and (B) immunodetection of proteins blotted on Immobilon-P (Millipore) membrane using mAb anti-MPII of gut supernatants from control and acute Cd-exposed worms (20 mg l–1, 2 days). Lane 3, molecular mass markers (kDa): 14.4, -lactalbumin; 20, trypsin inhibitor; 30, carbonic anhydrase; 43, ovalbumin; 67, albumin; 94, phosphorylase. Immunodetection of proteins from chromatographic fractions (see text) of gut supernatants revealed a main band of molecular mass <14.4 kDa and of similar intensity in control (lane 4) and Cd-exposed worms (lane 5).

View larger version (50K): [in a new window]   Fig. 3. Immunocytochemical characteristics of MPII-containing cells (MPII-C). (A) Anti-MPII immunoreactive cells (MPII-C) in the intestine of a control worm. Scale bar, 50 µm. (B) Immunoreactive MPII-C showing the characteristic nucleolus (arrow). l, gut lumen. Scale bar, 10 µm. (C) Immunoreactive cells (arrow) in the intestine of the sipunculid Sipunculus nudus. l, gut lumen; ep, epithelium; m, muscles. Scale bar, 50 µm.

View larger version (90K): [in a new window]   Fig. 4. In situ hybridization of MPII mRNA in intestine of control worms. (A) Labelling obtained using a digoxygenin anti-sense riboprobe. Note the labelling is specific to the MPII cells. l, lumen; c, coelom. (B) Using the sense riboprobe, no labelling was detected. Scale bar, 10 µm.

View larger version (163K): [in a new window]   Fig. 5. Ultrastructural characteristics of MPII-containing cells (MPII-C). (A) Control worm intestine. Numerous mitochondria (m) are dispersed in the cytoplasm. ER and Golgi apparatus are poorly developed. nu, nucleolus. (B) MPII-C of the intestine from a chronically Cd-exposed worm, showing characteristic granules (g). mf, micro-filaments. (C) MPII-C from an acute Cd-exposed worm. Note the development of endoplasmic reticulum (ER) and Golgi apparatus (Go). Nucleolus (nu) shows intrication of fibrillar and granular material. En, enterocyte nucleus. (D) Proliferation of granular nucleolar material (asterisk) in MPII-C from a chronically Cd-exposed worm. Scale bar, 1 µm.

View larger version (12K): [in a new window]   Fig. 6. Results of the ELISA on midgut extracts using the mAb anti-MPII. (A) Control worms, (B) chronically Cd-exposed worms, (C) acute Cd-exposed worms. Values are means ± S.E.M.. (N=3) ***Significant difference (P<0.001).

View larger version (38K): [in a new window]   Fig. 7. Semi-quantitative northern blot analysis: MPII mRNA levels were unchanged regardless of intoxication in control (A), acute (B) or chronically (C) intoxicated worms. (A) Northern blot of 18S and MPII RNA (see Materials and methods). (B) Density quantification of MPII RNA content after analysis using Quantity One software (Bio-Rad), relative to 18S expression level.