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.

Life-cycle exposure to cadmium induced compensatory responses towards oxidative stress in the liver of female zebrafish.

The current study examined effects of waterborne cadmium (Cd) at environmental relevant concentrations (0, 2.5 and 5 µg/L) on growth, survival, histology, ultrastructure, metal homeostasis, and antioxidant responses in female zebrafish from embryos to sexually maturity for 15 weeks. Growth and survival rate were not significantly affected by Cd exposure. There were no significant changes in ultrastructure of cellular organelles, reactive oxygen species (ROS) levels, lipid peroxidation (LPO) in liver. However, Cd exposure increased Cd and lipid accumulation, reduced contents of zinc, copper and reduced glutathione (GSH), and down-regulated activity of copper/zinc-superoxide dismutase (Cu/Zn-SOD) in liver of zebrafish. Contrarily, the mRNA and activity levels of catalase (CAT), the mRNA levels of Cu/Zn-SOD, and the mRNA and protein levels of metallothioneins (MTs) were up-regulated. The transcriptional regulation of Cu and Zn transporters might be a vital mechanism by which fish slow the Zn and Cu uptake. Taken together, our data demonstrated that long-term and low-dose Cd induced adaptive responses with interlinked compensatory mechanism, which may protect fish against oxidative stress.

Positive and negative innate immune responses in zebrafish under light emitting diodes conditions.

Certain light emitting diodes (LEDs) have become popular in fish farming beacause of a promoting effect on growth and reproduction. However, little information is available on innate immune responses in related tissues under LEDs conditions. The present study assessed the effects of a white fluorescent bulb (the control) and two different light-emitting diodes (LEDs: blue, LDB, peak at 450 nm; red, LDR, 630 nm) on growth and innate immune responses in the serum, liver and ovary of zebrafish for 8 weeks. LDB significantly enhanced specific growth rate (SGR), food intake (FI), and serum globulin levels. In contrast, LDR sharply inhibited SGR, FI, and the levels of albumin and globulin. Under LDB condition, there was an increase in protein levels of alkaline phophatase (AKP) and protein and activity levels of lysozyme (LZM) in the liver, and the levels of mRNA, protein, and activity of LZM in the ovary. Under LDR condition, LZM was dramatically down-regulated at mRNA, protein and activity levels in the ovary, suggesting that LZM was regulated at a transcriptional level. In the liver of the LDR group, though AKP mRNA levels sharply increased, its protein and activity levels significantly declined, indicating that AKP was regulated at translational level. Furthermore, a positive correlation between transcription factor NF-κB RelA mRNA levels and expression levels of AKP and LZM was observed in the liver and ovary, implying a transcriptional regulation of NF-κB RelA. In conclusion, the present study demonstrated a positive effect of LDB and negative effect of LDR on fish growth and innate immune responses, possibly associated with modifications at transcriptional, translational, and post-translational levels, and the transcriptional regulation of the NF-κB signaling molecule.

Chronic waterborne zinc and cadmium exposures induced different responses towards oxidative stress in the liver of zebrafish.

Based on the same toxic level of 0.6% LC50 for 96-h and the severe situation of water pollution, we compared effects of chronic Zn (180µgL(-1)) and Cd exposures (30µgL(-1)) on growth, survival, histology, ultrastructure, and oxidative stress in the liver of zebrafish for 5 weeks. Growth performance and survival rate remained relatively constant under Zn stress, but was reduced under Cd exposure. Cd exposure also induced severe pyknotic nuclei, evident ultrastructure damage, and considerable lipid inclusions in the hepatocytes. However, these phenomena were not pronounced under Zn exposure. The negative effects caused by Cd may be explained by an increase in hepatic oxidative damage, as reflected by the enhanced levels of lipid peroxidation (LPO) and protein carbonylation (PC). The reduced activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and catalase (CAT) may result in the enhanced hepatic oxidative damage, though the mRNA and protein levels of both genes increased and remained unchanged respectively. On the contrary, Zn up-regulated the levels of mRNA, protein and activity of Cu/Zn-SOD, which may contribute to the decreased LPO levels. Nonetheless, the sharply up-regulated mRNA levels of CAT did not induce an increase in the protein and activity levels of CAT under Zn stress. Furthermore, transcription factor NF-E2-related factor 2 (Nrf2) expression parelleled with its target genes, suggesting that Nrf2 is required for the protracted induction of antioxidant genes. In conclusion, our data demonstrated that essential and non-essential metals induced some differences in oxidative damage in fish. The differences were not caused by the transcriptional level of related genes but depended on post-transcriptional modifications.

[Effects of thymosin alpha1 on immune effector molecules of mouse].

OBJECTIVE: To analysis the effects of Talpha1 on the immune effector molecules in mouse immune system. METHODS: Sixty five BABL/c mice were divided into four groups: CsA group (n=20), Talpha1 group (n= 20), CsA+Talpha1 group (n=20) and control group (n=5). In the 3 experimental groups, 10 mg/kg CsA, 400 microg/ kg Talpha1, 10 mg/kg CsA+400 microg/kg Talpha1 were respectively administrated by intraperitoneal injection daily. Luminex was performed for cytokine detection at 1 d, 7 d, 14 d, 21 d day after the above treatments. Lymphocyte culture was prepared with the mouse spleen suspension, and then treated with 0. 25 mg/mL CsA, 10 microg/mL Talpha1 or 0.25 mg/mL CsA+10 microg/mL Talpha1 in vitro, respectively. Three days later, OD values of each treated lymphocyte culture and several cytokines in the culture were measured. RESULTS: Compared with other groups, CsA+Talpha1 group had significant lower IL-1alpha, IL-2, IL-6, IL-17, and significant higher IL-10 at 1 d, 7 d, 14 d, 21 d after the treatments (P < 0.05). Three days after the culture, OD value in the control group was significantly higher than that in Talppha1 group, CsA group, and CsA+ Talpha1 group (P < 0.05). IL-1alpa and IL-6 in the control group were significantly higher than those in the experiment groups (P < 0.05), while IL-10 in the control group was significantly lower than that in the experiment groups (P < 0.05). IL-2 and IL-17 were similar. CONCLUSION: Talpha1 show regulatory effect on the immune effector molecules which could promote Th1 cells transforming to Th2 cells.

[Establishment of full-sib families of Branchiostoma japonicum and the relationship between early development patterns and larvae survival rates].

One general requirement of individual laboratory animals is that they have known genetic backgrounds. However, ensuring such genetic similarity is difficult, and can be facilitated by breeding a full strain for experimentation. To this end, the authors bred 34 full-sib families of amphioxus larvae/embryos. Due to the high mortality of the embryos and larvae, only seven full-sib families of juvenile amphioxus Branchiostoma japonicum were obtained. Among them, the highest and lowest survival ratios were 32.4% and 1.67%, respectively, whereas the shortest metamorphosis and longest larva duration were 24 d and 42 d, respectively. These results demonstrate the feasibility of establishing full-sib families of amphioxus, and provide fundamental data needed for the future breeding of amphioxus strains.

Axonal loss and blood flow disturbances in the natural course of indirect traumatic optic neuropathy.

BACKGROUND: Indirect traumatic optic neuropathy (TON) is an acute injury of the optic nerve associated with severe visual dysfunction, which may be a result of secondary mechanical injury and vascular disorder of the optic nerve due to trauma. We analyzed the natural course of axonal loss and blood flow disturbances in patients with indirect TON to find a possible therapeutic window. METHODS: A cohort of 54 patients with indirect TON recruited between October 2008 and October 2010 at Beijing Tongren Hospital was retrospectively analyzed. The patients were divided into no light perception group (NLP) and better than NLP (btNLP) group. Specifically, the thickness of the retinal nerve fiber layer (RNFL) measured by spectral domain optical coherence tomography (SD-OCT), and hemodynamic parameters of the ophthalmic artery (OA), central retinal artery (CRA) and posterior ciliary artery (PCA) were determined. RESULTS: Two weeks after injury, there was a statistically significant decrease in the thickness of RNFL in the btNLP group as compared with the fellow control eyes (P < 0.05). In contrast, in the NLP group, RNFL thickness slightly increased for 2 weeks following injury, then overtly reduced after 4 weeks (P < 0.05). Peak systolic velocity (PSV) of CRA was significantly decreased 4 weeks after injury (P < 0.05) in both the NLP group and btNLP group (P < 0.05). The thickness of RNFL in the NLP group was negatively correlated with PSV of CRA after 1 week of injury (P < 0.05, r = -0.962). CONCLUSIONS: SD-OCT is a useful supplement in detecting the axonal loss in TON. The dynamic change of the thickness of RNFL appears to correlate with the hemodynamic disturbances in the natural course of TON. The first 2 weeks following an injury is critical and should be considered as the therapeutic window for TON patients.

[Identification and expressional analysis of green fluorescent protein genes in amphioxus].

Endogenous green fluorescent proteins (GFPs) have recently been found in amphioxus, where different characteristics of light production in different development stages and between different individuals have manifested. We identified the GFP genes in an amphioxus species Branchiostoma belcheri and monitored real time fluorescence signals of GFPs during different developmental stages to provide an insight on the GFP function in amphioxus. We found there are at least 12 endogenous GFP genes in amphioxus genome, and fluorescent expression changes in body position during different developmental stages. Additionally, GFP expression after metamorphosis development differed significantly among different amphioxus individuals, suggesting that there are multiple GFP homologous genes responsible for fluorescent expression. Expression levels of these genes varied significantly during different developmental stages, indicating that different GFP genes may have their unique functions in the development of amphioxus during specific phases of growth.

Erythropoietin receptor positive circulating progenitor cells and endothelial progenitor cells in patients with different stages of diabetic retinopathy / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the possible involvement of erythr opoietin (EPO)/erythropoietin receptor (EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR).</p><p><b>METHODS</b>EPOR positive circulating progenitor cells (CPCs: CD34(+)) and endothelial progenitor cells (EPCs: CD34(+)KDR(+)) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients with out diabetes ( n=7),non-proliferative DR (NPDR, n=7),non-proliferative DR (PDR, n=8), and PDR complicated with diabetic nephr opathy (PDR-DN, n=7).</p><p><b>RESULTS</b>The numbers of EPOR(+) CPCs and EPOR(+) EPCs were reduced remarkably in NPDR compared with the control group (both Pü0.01), whereas rebounded in PDR and PDR-DN groups in varyingdegrees. Similar changes were observed in respect of the proportion of EPOR(+)CPCs in CPCs (NPDR vs. control, Pü0.01) and that of EPOR(+) EPCs in EPCs (NPDR vs. control, Pü0.05).</p><p><b>CONCLUSION</b>Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the impaired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR(+)EPCs associated with ischemia.</p>

[Analysis of amphioxus geographic populations in the West Pacific Ocean based on COX I gene].

Amphioxus (lancelet) is an important model animal in the field of Evo-Devo study. However, the specimens for laboratorial experiments are usually sampled directly from the field. Thus, it is necessary to correctly identify species and evaluate the genetic diversity of geographic populations. In the present investigation, we amplified and sequenced COX I gene of amphioxus collected from various localities of the West Pacific Ocean and downloaded all available sequences of the gene from NCBI. Based on those sequences data, we constructed NJ tree to decide the taxonomic status of those amphioxus and performed AMOVA analysis to estimate genetic diversities among geographic populations and individuals. The current results supported the validity of the three amphioxus species, Branchiostoma malayanum, B. belcheri and B. japonicum. We also proposed that some amphioxus species names recorded in NCBI sequence database might be inaccurate. Genetic analysis on the geographic populations of amphioxus species suggested that each examined amphioxus species have a high level of genetic diversity. However, the genetic divergences among geographic populations of the same species are indetectable, indicating a frequent gene flow among them.

Pharmacokinetic and toxicity study of intravitreal erythropoietin in rabbits.

AIM: To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use. METHODS: For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10 U, 100 U, or 1,000 U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6X). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d. CONCLUSIONS: IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit.