Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide.

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1h), including alpha-2-macroglobulin (alpha2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by alpha-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.

Cloning the prophenoloxidase cDNA and monitoring the expression of proPO mRNA in prawns (Macrobrachium rosenbergii) stimulated in vivo by CpG oligodeoxynucleotides.

The prophenoloxidase (proPO) system forms the basis of the non-specific defence system in crustaceans. The aim of this study was to develop an RT-PCR procedure to determine proPO gene expression. We used several degenerate primers designed from the conserved regions in amino acid sequences of proPOs from other species to clone the possible cDNA(s) from the haemocytes of the prawn Macrobrachium rosenbergii. One DNA fragment, 2016 bp long, was cloned by a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (3'/5'-RACE). This fragment could encode a putative polypeptide with 671 amino acids. Further analysis showed that this putative polypeptide contained six histidine residues and a thiol ester-like motif (GCGWPRHM) just like the structural features of proPOs from the shrimp Penaeus monodon. A partial fragment of the sequence with 934 bp containing three histidine residues and the thiol ester-like motif was used as a target to monitor the activation of the proPO gene by the semi-quantitative RT-PCR analysis. The result showed that the highest level of proPO mRNA was detected at 1h after in vivo injection with 5 microg of CpG oligodeoxynucleotide 2006 per prawn; in the same experiment, the highest PO activity was detected at 6 h after injection. In the control, a continuous and slow elevation of PO activity was observed during the experimental period, but such elevation of proPO mRNA was not observed. From our previous study and the time course of gene expression of proPO and enzymatic activity of PO in this study, it can be concluded that the enhancement effect was through its transcriptional level, its translational level and then its post-translational level sequentially. These results suggest that, both for reliability, sensitivity and for the timing of sampling, the change in proPO mRNA is more useful than the PO activity in monitoring the activation of the proPO non-specific defence system of prawns after treatment with stimulants.

Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells.

Phthalates are widely used in industry and cause public concern since they have genomic estrogenic-like effects via estrogen receptors. We previously found that some phthalates have nongenomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChRs) in bovine chromaffin cells. In this study, we investigated the effects of eight phthalates on the calcium signaling of human nAChR by using human neuroblastoma SH-SY5Y cells. All eight phthalates, with different potency, have inhibitory roles on the calcium signaling coupled with human nAChR, but not muscarinic acetylcholine receptors (mAChRs). For inhibition of human nAChR, the strongest to weakest potencies were observed as di-n-pentyl phthalate (DPP) --> butyl benzyl phthalate (BBP) --> di-n-butyl phthalate (DBP) --> dicyclohexyl phthalate (DCHP) --> di-n-hexyl phthalate (DHP) --> di-(2-ethyl hexyl) phthalate (DEHP) --> di-n-propyl phthalate (DPrP) --> diethyl phthalate (DEP). The potencies of phthalates were associated with their structures such that the most effective ones had dialkyl group carbon numbers of C4 or C5, with shorter or longer numbers resulting in decreased potency. At as low as 0.1 microM, DPP, DBP, BBP, DCHP and DHP significantly inhibited the calcium signaling of human nAChR. The IC50 of phthalates on human nAChR, ranging from 0.32 to 7.96 microM, were 10-50 lower than those for bovine nAChR. We suggest that some phthalates effectively inhibit the calcium signaling of human nAChR, and these nongenomic effects are cause for concern.