Guangwen Chen

Cloning and expression analysis of hsp70 gene from freshwater planarian Dugesia japonica

Heat shock protein 70 (HSP70) is an important member of the heat shock protein family. It plays a key role in the process of protecting cells by facilitating the folding of nascent peptides as well as the cellular stress response. We isolated and sequenced a full-length HSP70 cDNA from planarian Dugesia japonica (designated Djhsp70) using rapid amplification of cDNA ends (RACE). Djhsp70 cDNA is highly homologous to other reported hsp70 family genes, and the deduced amino acid sequence shares several eukaryotic HSP70 family motifs. The length of the Djhsp70 ORF from planarian genomic DNA and cDNA was identical, which indicated the absence of introns in the Djhsp70 gene. Semi-quantitative RT-PCR was employed to detect the expression levels of Djhsp70 in response to stressors. Our results indicated that Djhsp70 was an inducible gene, expressed in response to temperature changes, amputation and starvation. Interestingly, the phylogenetic analysis of DjHSP70 supports the idea that planarians belong to a new phylogenetic position — Lophotrochozoans. This is the first molecular analysis of a heat shock protein gene in planarians.

Molecular characterization of the glucose-regulated protein 78 (GRP78) gene in planarian Dugesia japonica

GRP78 (78 kDa glucose-regulated protein) has ubiquitously existed in nearly all organisms from yeast to humans, reflecting the central roles it plays in cell survival. In this report, we isolated and sequenced the full-length cDNA of GRP78 (designated DjGRP78) from the planarian Dugesia japonica. The cDNA is 2121 bp, including an open reading frame (ORF) of 1983 bp encoding a polypeptide of 660 amino acids with three HSP70 family signatures. DjGRP78 contains signal peptides at the N-terminus and a KTEL peptide motif at the C-terminus, which suggests that it localizes in the endoplasmic reticulum (ER). Fluorescent real time RT-PCR was employed to detect the expression pattern of Djgrp78 in response to different stressors. Our results show that heat shock and heavy metals (Hg(2+) and Pb(2+)) induce Djgrp78 expression, but starvation does not. Interestingly, we found that Djgrp78 was up-regulated in planarians with septic tissues, and also verified that it was up-regulated in response to bacterial challenge. Our data indicate that Djgrp78 may be a multifunctional gene, and play important roles in physiological and pathological stress in planarians.

Photokinesis and Djopsin gene expression analysis during the regeneration of planarian eyes.

Planarians provide the ideal model for studying eye development, with their simple eye structure and exceptionally rapid regeneration. Here, we observed the eye morphogenesis, photophobic behavior, spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica. The results showed that: (i) Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution; (ii) planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue (495 nm) and yellow (635 nm); (iii) the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration; and (iv) Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians.

Genotoxicity evaluation of ionic liquid 1-octyl-3-methylimidazolium bromide in freshwater planarian Dugesia japonica using RAPD assay.

The randomly amplified polymorphic DNA (RAPD) assay has been used to detect DNA alternation and mutation recently. However, the effectiveness of this method in detecting DNA damage in planarians, a model organism for assessing the toxicity of environmental pollutants is unknown. In the present study, RAPD assay was used to detect the DNA damage in planarians treated by the ionic liquid 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for the first time. Among the 20 test RAPD primers, 13 primers with 60-70% GC content produced unique polymorphic band profiles. A total of 60 bands were observed in the untreated control planarians. In comparison with the control group, the [C8mim]Br-treated groups displayed differences in RAPD patterns in the band intensity, disappearance of normal bands and appearance of new bands. The variation of RAPD profiles showed both concentration- and time-effect relationships. Meanwhile, the genomic template stability (GTS) of treated planarians decreased and exhibited negative correlation to the exposure concentration and time of [C8mim]Br. Our results suggested that [C8mim]Br had genotoxic effects on planarians, and this DNA damage analysis would lay the foundation for further elucidating the toxicity mechanisms of ionic liquids on planarians. Furthermore, RAPD analysis was proved to be a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals on planarians.

Identification and expression analysis of a heat-shock protein 70 gene in Polycelis sp.

Heat-shock protein 70 (HSP70) is ubiquitously found in a variety of organisms and plays an important role in cytoprotection, environmental monitoring, and disease resistance. In this study, the full-length complementary DNA (cDNA) of hsp70 from planarian Polycelis sp. was first cloned using rapid amplification of cDNA ends (RACE). The expression levels of Pyhsp70 were analyzed in the presence of various stressors by real-time PCR, and its temporal-spatial expression patterns were also examined in both intact and regenerative animals by whole-mount in situ hybridization. The results show that (1) the deduced amino acid sequence of Pyhsp70 includes three typical HSP70 family signature motifs and is highly conserved during evolution; (2) Pyhsp70 expression is induced by prolonged starvation, tissue damage, and ionic liquid but inhibited by high or low temperatures; and (3) Pyhsp70 mRNA is mainly expressed in the head peripheral region and in the regenerating blastema during regeneration. These results suggest that the highly expressed Pyhsp70 gene may contribute to enhance cytoprotection and tolerance against stress-induced molecular damage, and the migration of neoblasts to the wound, which might also be involved in the proliferation and differentiation of neoblasts. Our work provides basic data for the study of stress responses and regenerative mechanism in freshwater planarians.

Genotoxicity Evaluation of an Urban River on Freshwater Planarian by RAPD Assay

The aim of this study was to evaluate the genotoxic potential of an urban river – the Wei River in Xinxiang, China using randomly amplified polymorphic DNA (RAPD) assay in planarians. The results showed that the total number of polymorphic bands and varied bands in RAPD patterns of treated planarians decreased with the water sample site far away from the sewage outlet of a factory. In addition, the genome template stability of treated groups decreased and the degree of the decline was negatively related to the distance between the sample site and the sewage outlet, suggesting that the Wei River water had genotoxicity effects on planarians and strengthening the management of the Wei River was necessary. Furthermore, this work also indicated that RAPD assay in planarians was a very promising test for environmental monitoring studies.

Changes of alkaline phosphatase activity in response to different stressors in planarian Dugesia japonica

The aims of this work are to provide some properties of alkaline phosphatase (ALP) in the planarian Dugesia japonica and detect its activity in response to different stressors, as well as to introduce renatured SDS-PAGE to study enzyme activity in planarians. Our results indicate that ALPs in planarians are mainly membrane-bound form, identified as three mainly enzyme-bands (approximately MW 260 kD, 180 kD, 160 kD, respectively). Under our experimental conditions, ALPs activity had no apparent changes in response to low concentration of Hg2+ (25 μg L−1) and Pb2+ (125 μg L−1, 250 μg L−1) exposure, but were severely inhibited in response to high concentration of Hg2+ (50 μg L−1, 150 μg L−1, 300 μg L−1) and Pb2+ (500 μg L−1, 1000 μg L−1) exposure. Mild heat shock (25°C for 2 days) elevated ALP activity, but severely heat shock (25°C for 2 days, followed by 30°C for 2 days and 32°C for 2 days) inactivated ALP activity. Interestingly, ALP and other cytosolic phosphatases (MW from ∼45 kD to ∼60 kD) activity increased noticeably during the early stage of planarians regeneration, which may be involved in cell proliferation and differentiation. Contrary to regeneration, prolonged starvation suppressed ALP activity. The above findings provide valuable information about the role of ALP in planarian regeneration and for its use as an indicator in ecotoxicology.

Identification of a HSP40 gene involved in planarian regeneration

Abstract Heat shock protein 40 (HSP40), as an essential co-chaperone of HSP70, has been poorly investigated in planarians. In this study, a full-length cDNA of a HSP40 gene from planarian Dugesia japonica (designated Djhsp40) was identified and its expression pattern during planarian regeneration was examined. Its cDNA is 1,378 bp, which includes an open reading frame of 1,236 bp encoding 411 amino acids with a molecular weight of 46.2 kDa. DjHSP40 contains three conserved DNAJ domains belonging to the Type I HSP40/DNAJ family. After amputation is performed, Djhsp40 is up-regulated and the transcripts are specifically expressed in the blastema, and its inhibition by siRNA strongly delays the blastema growth. Interestingly, the silencing of Djhsp40 does not affect the expression level of neoblast-specific proliferation marker (Dj-mcm2 and DjpiwiB), but reduces Dj-mt-mmpA expression significantly. Our work provides a crucial clue for the study of the function of HSP40 in planarian regeneration.

Identification and expression analysis of a Spsb gene in planarian Dugesia japonica

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb. The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1, Spsb2 and Spsb4; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians.

Identification of runt family genes involved in planarian regeneration and tissue homeostasis

The runt family genes play important roles in physiological processes in eukaryotic organisms by regulation of protein transcription, such as hematopoietic system, proliferation of gastric epithelial cells and neural development. However, it remains unclear about the specific functions of these genes. In this study, the full-length cDNA sequences of two runt genes are first cloned from Dugesia japonica, and their roles are investigated by WISH and RNAi. The results show that: (1) the Djrunts are conserved during evolution; (2) the Djrunts mRNA are widely expressed in intact and regenerative worms, and their expression levels are up-regulated significantly on day 1 after amputation; (3) loss of Djrunts function lead to lysis or regeneration failure in the intact and regenerating worms. Overall, the data suggests that Djrunts play important roles in regeneration and homeostatic maintenance in planarians.