A functional gene encoding carnitine palmitoyltransferase 1 and its transcriptional and kinetic regulation during fasting in large yellow croaker.

Carnitine palmitoyltransferase 1 (CPT1) plays an essential role in maintaining energy supply via fatty acid oxidation, especially under fasting. In this study, the complete cDNA sequence of cpt1a was cloned from liver of large yellow croaker (Larimichthys crocea), with an open reading frame of 2319 bp encoding a protein of 772 amino acids. Bioinformatics analysis predicted the presence of conserved functional motifs and amino acid residues. The highest mRNA expression of cpt1a was observed in the liver. Phylogenetic tree clearly shows that CPT1A protein is a homologue of mammalian CPT1A. Recombinant protein rCPT1A showed catalytic activity, with Michaelis constant (km) (≈1.38 mM) and maximal reaction rates (Vmax) for carnitine (≈12.66 nmols/min/mg protein). The cpt1a mRNA expression dramatically increased and CPT1 activity remained unchanged after fasting. Fasting did not significantly change Vmax and free carnitine (FC) content in liver. Interestingly, catalytic efficiency (Vmax/Km) and FC/Km increased in fish fasted for 4 days, implying FC contents might be enough to ensure the optimal fatty acid oxidation. Contrarily, both indicators declined when fish fasted for 12 days. The present results demonstrated cpt1a has a biological function and showed that the transcriptional and kinetic regulation of CPT1 during fasting, emphasizing that fasting-induced fatty acid oxidation depends on changes in kinetic properties instead of CPT1 activity and transcription.

Negative effects of acute cadmium on stress defense, immunity, and metal homeostasis in liver of zebrafish: The protective role of environmental zinc dpre-exposure.

In the study, zebrafish were exposed to 0 and 200 µg/L Zn for 8 weeks, and then both groups were transferred to water including 0, 100, and 200 µg/L Cd for 4 days, respectively. Acute Cd exposure caused negative effects on stress defense, immune, and metal transport systems by increasing lipid peroxidation, iNOS activity and mRNA levels of il-6 and inos, and decreasing Cu/Zn-SOD and HSP70 levels, and mRNA levels of sod1, cat, hsp70, p65, mtf-1, znt5, zip7, atp7a, and atp7b. Lipid peroxidation was significantly reduced by Zn pre-exposure under Cd exposure, which may be explained by the enhanced stress defense capacity and the weaken inflammatory response. Firstly, Zn pre-exposure increased MTs and HSP70 levels and CAT activity in Cd-free water, which may facilitate fish quick response to Cd. Secondly, Zn pre-exposure reduced Cd accumulation at 100 and 200 µg/L Cd, down-regulated il-6 and il-1ß at 100 µg/L Cd and p65 at 200 µg/L Cd, and increased Cu/Zn-SOD and CAT activities at 200 µg/L Cd. Thirdly, Zn pre-exposure alone up-regulated transcription factors (hsf1, hsf2, and mtf-1, and nrf2) and their target genes (sod1, cat, hsp70, and mt2) under Cd exposure in a dose-dependent manner. It should be noted that Zn pre-exposure down-regulated several metal transport genes dramatically at 0 and 100 µg/L Cd, which may be an important mechanism for reducing Cd import into livers. Overall, long-term and environmental Zn pre-exposure mitigated Cd toxicity by the enhanced stress defense capacity and the down-regulated metal transport and inflammatory responses.

The lagged effects of environmentally relevant zinc on non-specific immunity in zebrafish.

Responses to zinc (Zn) during exposure have well studied but the effects after the exposure are commonly neglected. In the study, non-specific immune response to zinc in blood and spleen of zebrafish was evaluated after exposure. At first, fish were subjected to 0 (control) and 200 µg/L zinc (Zn) for 6 weeks. Specific growth rate, survival rate, blood albumin level, and the activities of Cu/Zn-SOD and iNOS were not significantly changed by Zn exposure. Conversely, Zn increased the levels of globulin and hemoglobin, CAT activity, and mRNA levels of nrf2, sod1, cat, hsf1, hsp70, p65, il-6, il-1ß, tnf-α and inos. In the second experiment, zebrafish were transferred to a recovery period for 4 and 8 days. The increased activities of Cu/Zn-SOD and CAT and the up-regulated mRNA levels of nrf2, cat, p65, tnf-α, and inos still were observed. In the third experiment, zebrafish from 4 d post-exposure were re-exposed to the high levels of Zn and cadmium (Cd) (600, 1200 µg/L Zn; 100, 200 µg/L Cd) for 4 days. 100 µg/L Cd caused a higher survival rate in the Zn-exposed fish than the control, suggesting Zn pre-exposure might develop the tolerance to Zn and Cd. Although transcriptional levels of sod1, hsf1, hsf2, hsp70, il-6 and il-1ß and activity levels of iNOS recovered to the control levels at 4 and 8 d post-exposure, differences in magnitude of responsiveness were observed between normal fish and Zn-exposed fish. Overall, Zn acclimation persisted when fish recovered, which provides a new perspective about Zn toxicology.

Transcriptional and physiological responses of Dunaliella salina to cadmium reveals time-dependent turnover of ribosome, photosystem, and ROS-scavenging pathways.

Effects on short-term (6 h) and long-term (96 h) exposure to cadmium (Cd) at 0.1, 0.5 and 2.5 mg/L in microalga Dunaliella salina were assessed using both physiological end points and gene expression analysis. Different physiological responses between the short-term and long-term exposures were observed. Upon 6 h after Cd exposure, lipid peroxidation and cell ultrastructure remained unchanged, while contents of chlorophyll a, chlorophyll b, carotenoids were increased at 0.5 and 2.5 mg/L Cd. Contrarily, 96 h after Cd exposure, lipid peroxidation levels were increased, while pigments content was decreased, and damaged cell ultrastructure was apparent at 2.5 mg/L Cd. Activities of antioxidant enzymes (APX, SOD, GST, GPX, and GR) changed differently both at 6 h and 96 h after Cd exposure. Upon 6 h after Cd exposure, SOD and GST activity increased at all three doses, GR and GPX activity increased at 0.5 mg/L Cd while APX activity increased at 0.1 mg/L Cd. Contrarily, 96 h after Cd exposure, activities of all the antioxidant enzymes increased both at 0.1 and 0.5 mg/L Cd; but there was a decrease in SOD and GR activity in D. salina exposed to 2.5 mg/L Cd. RNA-seq and qRT-PCR analyses indicated that genes involved in ROS-scavenge, photosystem, and ribosome functions were differentially expressed. The most significantly enriched function was the ribosome, in which more than 30 ribosome genes were up-regulated at 6 h but down-regulated at 96 h after Cd exposure at 2.5 mg/L. Our study indicated for the first time that genes encoding ribosomal proteins are the primary target for Cd in microalgae, which allowed gaining new insights into temporal dynamics of toxicity and adaptive response pathways in microalgae exposed to metals.