Characterization of the tilapia p53 gene and its role in chemical-induced apoptosis.

Roles of p53 in mammals have been extensively studied but little is known about its functions in lower vertebrates. We have cloned and characterized the p53 gene from tilapia (Oreochromis niloticus), expressed it in Escherichia coli, generated a rabbit polyclonal antibody and examined the gene's expression patterns at both transcript and protein levels. The full-length p53 cDNA was 1288 bp with an ORF of 1,138 bp, encoding a polypeptide of 364 amino acids. The gene contains all four classical p53 family signature motifs but has low sequence similarity with other p53 genes. It was constitutively transcribed in all tissues examined, most abundantly in liver and blood cells. In addition, ATM, p53 and Bax proteins, associated with DNA damage responses and apoptosis, were all upregulated in tilapia liver after treatment with the stress inducers etoposide and CdCl(2). Tilapia p53 is thus both a constitutive and inducible acute-phase protein that is cooperatively activated via the ATM-p53 pathway in responses to stress and DNA-damaging signals.

Molecular characterization and expression analysis of elongation factors 1A and 2 from the Pacific white shrimp, Litopenaeus vannamei.

Elongation factors (EF) are abundant cell proteins that play important roles in the metabolism of all multicellular organisms. Here we describe a functional analysis of elongation factor 1-alpha (EF1A) and elongation factor 2 (EF2), from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNAs of genes corresponding to EF1A and EF2 were obtained that were 1547 and 2729 bp long, with open reading frames encoding 461 and 846 amino acids, respectively. The deduced amino acid sequences of L. vannamei EF1A and EF2 showed high similarity with those from mice, humans, chickens and other shrimps. RT-PCR analysis indicated that mRNA transcripts of EF1A and EF2 are strongly (but differentially) expressed in haemocytes and gill tissue, and at varying levels in other examined tissues, of the shrimps. Levels of both EF1A and EF2 transcripts increased when shrimps were challenged by pH and cadmium stress, but reached maximal levels after different exposure periods. These results indicate that EF1A and EF2 may play distinct, essential roles in the repair of cellular damage induced by pH and cadmium stress.

Acute acidic exposure induces p53-mediated oxidative stress and DNA damage in tilapia (Oreochromis niloticus) blood cells.

Acid rain and inputs of acidic effluent can result in increased acidity in aquatic ecosystems, where it is known to have a significant impact and possibly, to cause the decline of some populations of aquatic organisms. In previous studies, intracellular acid-induced oxidative stress has been shown to cause DNA damage, and cooperatively activate the expression of the p53 gene. The acute effects of acidic environments on shrimp and fish have been widely studied. However, the molecular mechanism of acid-induced injury remains largely unknown. In this study, we examined the cellular responses of tilapia to acidic exposure-induced oxidative stress and antioxidant enzyme gene expression. Furthermore, we determined how acute acid stress activates the ATM-p53 signal pathway. We measured the upregulation of reactive oxygen species (ROS) production, the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)), the tail DNA values, the malondialdehyde (MDA) level in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in tilapia in conditions where the pH was 5.3. Apoptosis was detected by Hoechst staining, which was mainly associated with changes in cell viability. The parameters that we measured were related to acid-induced DNA damage, and all parameters changed in the blood cells through time. The effects of acute acid exposure (pH 5.3) on the expression of ATM, p53, p21, Bax, manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) were investigated in tilapia blood cells. The results showed that acute acid stress induced upregulation of ATM, p53 and p21, associated with increasing of DNA damage and apoptosis in blood cells. Additionally, the expression of Bax was slightly increased. Moreover, consensus p53-binding sequences were identified in tilapia MnSOD and GPx gene promoter regions and increased levels of ROS in the blood cells coincided with increased mRNA expression of p53, MnSOD and GPx. Therefore, it suggests that acid exposure-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively activate ATM-p53 pathway, which may lead to the activation of p21 and regulate transcription of MnSOD and GPx.

Protection of blue shrimp (Litopenaeus stylirostris) against the White Spot Syndrome Virus (WSSV) when injected with shrimp lysozyme.

In this study we found that a blue shrimp (Litopenaeus stylirostris) lysozyme gene (Lslzm) was up-regulated in WSSV-infected shrimp, suggesting that lysozyme is involved in the innate response of shrimp to this virus. Shrimp were intramuscularly injected with Lslzm protein to identify how this recombinant protein protects L. stylirostris from WSSV infection and to determine how this protein influences nonspecific cellular and humoral defense mechanisms. Higher survival rates and a lower viral load (compared with controls) were reported for shrimps that were first injected with the Lslzm protein and then infected with WSSV. In addition, the Lslzm expression level and the immunological parameters (including THC, phagocytic activity, respiratory burst activity, phenoloxidase activity and lysozyme activity) were all significantly higher in the WSSV-infected shrimp treated with the Lslzm protein, compared with the controls. These results indicate that lysozyme is effective at blocking WSSV infection in L. stylirostris and that lysozyme modulates the cellular and humoral defense mechanisms after they are suppressed by the WSSV virus.

Oxidative stress, DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp, Litopenaeus vannamei when exposed to acute pH stress.

The ROS production, the percentage of dead and damaged haemocytes, the DNA Olive Tail Moment (OTM) value and the gene expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx), were studied in the Pacific white shrimp, Litopenaeus vannamei, when exposed to acute pH stress. The increased ROS production in haemocytes and the increased OTM value in both the haemocytes and the hepatopancreas cells suggest that oxidative damage occurred in shrimp exposed to pH 5.6 and pH 9.3, with apoptosis, mainly being associated with excess Ca(2+)influx and changes in cell viability. Acid and alkaline pH-induced DNA damage was time dependent in the haemocytes and the hepatopancreas cells. The concentration of intracellular free calcium [Ca(2+)] (i) after different pH treatments increased significantly over time, reaching its highest concentration after 12 h, but decreasing gradually to normal levels after 24 h. The [Ca(2+)] (i) content in shrimp cells when exposed to pH 9.3 and pH 5.6 for 12 h had increased by 58%-81%, compared with exposure to pH 7.4 (control). In addition, the gene expression of cMnSOD, CAT, GPx and TRx in the hepatopancreas of L. vannamei was induced by acid and alkaline pH stress, although there were differences in the expression response with respect to the duration of induction and the different pH treatments (acid or alkaline). Our results show that acidic or alkaline-induced oxidative stress may cause DNA damage, and cooperatively activate expression of CAT, GPx and TRx mRNA. Calcium ions appear to be important in mediating shrimp responses to pH stress.

Molecular cloning, characterization, expression and antibacterial analysis of a lysozyme homologue from Fenneropenaeus merguiensis.

The gene coding for lysozyme in banana prawn (Fenneropenaeus merguiensis) was cloned, sequenced and expressed in pET-32a vector. The deduced amino acid sequence of F. merguiensis lysozyme showed 37-93% similarity with the mouse, human, chicken, and tiger prawn counterparts. The lysozyme was purified to homogeneity and observed as a band of approximately 15 kDa in 12% SDS-PAGE. Semiquantitative RT-PCR analysis demonstrated that mRNA transcripts of lysozyme could be mainly detected in the tissues of hemocytes, gill, gonad and lymphoid organ of unchallenged shrimps, whereas the expression of lysozyme transcripts was increased in all the tested tissues after heat-killed Vibrio alginolyticus challenge. The temporal expression of lysozyme mRNA in hemolymph challenged by Micrococcus luteus and V. alginolyticus was both up-regulated and reached the maximum level at 8 and 16 h post stimulation, respectively, and then dropped back to the original level. Bacteriolytic activity of lysozyme against different bacterial cultures was determined by solid phase as well as turbidimetric assay. Lysis was obtained against gram positive and gram negative bacteria with strong inhibition against shrimp pathogens V. alginolyticus and V. parahemolyticus. In addition, the study of inhibition mechanism revealed that the antibacterial activity of lysozyme was a result of bactericidal effect.