Induction of gene expression of the chaperones <t-1>14-3-3 and HSP70 by PCB 118 (2,3',4,4',5-pentachloro<t0>biphenyl) in the marine sponge Geodia cydonium:novel biomarkers for polychlorinated biphenyls

Abstract

Polychlorinated biphenyls (PCBs) are ubiquitous industrial compounds found in almost every component of the terrestrial and marine ecosystem. Most of the PCB congeners bind to the aryl hydrocarbon receptor and in turn cause expression of stress response genes. Here we report for the first time that PCB 118 acts in the marine sponge Geodia cydoniurn as an inducer of 2 chaperones, the 14-3-3 protein(s) (a protein targeting molecule) and the heat shock protein HSP70 (a chaperone, primarily involved in folding of proteins). While the cDNA encoding the latter protein has been cloned previously, the 14-3-3 cDNA from sponges is reported in this study. The full-length cDNA clone of G. cydoniurn, GC14-3-3, has a size of 912 nucleotides (nt) and contains a 744 nt long potential open reading frame; the relative molecular weight (M,) of the deduced aa sequence is 28 378 Da. The sponge polypeptide is closely related to the deduced polypeptides of the 14-3-3 sequences belonging to isoforms q and y. Using the cDNAs, coding for the 14-3-3 and the HSP70 protelns as well as antibodies raised against these 2 proteins, it was demonstrated that neither chaperone can be detected in the absence of PCB. However, after incubation of sponge tlssue w t h PCB 118 the transcripts of the 2 chaperones are detectable after 12 h, while the corresponding proteins appear after 1 d . Subsequently, the levels of the transcripts and of the proteins increase steably. From these data we conclude that the 2 chaperones, 14-3-3 and HSP70, are useful biomarkers in sponges. Due to the broad cross-reactivity of their antibodies throughout the Metazoa, these chaperones may be useful biomarkers for monitoring environmental contaminants, as shown here for PCB 118, in all organisms.

Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293 cells

“…65,66 RNA (total) samples (5 mg) were electrophoresed in a formaldehyde/1% agarose gel and blotted onto Hybond N + membrane following the manufacturer's instructions (Amersham; Little Chalfont, Buckinghamshire; UK). Hybridization was performed with the 586 nt long GCBHP2 fragment (spanning nt 83 to nt 668 of the ORF) in HighNaDodSO 4 hybridization Buffer (7% NaDodSO 4 , 50% formamide, 56SSC, 0.1% Na-laurylsulfate, 50 mM Na 3 PO 4 [pH 7.0] and 2% of blocking reagent [Roche]) at 428C.…”

The 60-kDa Heat Shock Protein (HSP60) of the Sea Anemone Anemonia viridis: A Potential Early Warning System for Environmental Changes

“…These proteins act to protect the organism from cell protein damage due to exposure to a wide variety of stressors, including elevated temperature (Lindquist, 1986;Sanders, 1993), cold shock (Nunamaker et al, 1996), oxidative stress (Lindquist, 1986), increased UV irradiation (Sanders, 1993), extreme pH (Koziol et al, 1996), osmotic stress (Burg et al, 1996), heavy metals (Köhler et al, 1992;Miller et. al., 1992;Sanders, 1993;Vedel and Depledge, 1995), xenobiotics (Sanders, 1993), and other pollutants (Lindquist, 1986;Blom et al, 1992;Pomerai, 1996;Wiens et al, 1998).…”

Purification, cDNA cloning and expression of a cadmium-inducible cysteine-rich metallothionein-like protein from the marine sponge Suberites domuncula

“…The PCR reaction was carried out at an initial denaturation at 95°C for 3 min, then 30 amplification cycles at 95°C for 30 S, 52°C for 45 S, 74°C for 1.5 min, and a final extension step at 74°C for 10 min. The reaction mixture was as described earlier (Wiens et al 1998). The longest fragment of -500 bp was used and completed by primer walking as described by Ausubel et al (1995).…”

“…Studies revealed that sponges can cope with organic pollutants and organismic attacking invaders by (1) cellular defense systems, e.g. heat-shock proteins (Koziol et al 1997), targeting chaperones like the 14-3-3 protein(s) (Wiens et al 1998), multidrug resistance (MDR) (reviewed in Kurelec 1997) or thioredoxin (Wiens et al 1999) and by (2) organismic protection, including a well-developed immune system ) and bioactive secondary metabolites (Sarma et al 1993, Proksch 1994. Recently, we also identified the second MDR-system, related to the POHl Lpad one homologoue] mechanisms (Spataro et al 1997), which is thought to 'eliminate' pollutants via an enzymatic route (Krasko et al 2000).…”