Short communication: optimization of semi-quantitative RT PCR analysis for CPT I gene expression in rainbow trout (Oncorhynchus mykiss)

A key enzyme in mitochondrial β¬oxidation, carnitine palmitoyltransferase (CPT) I, is transcriptionally regulated in mammals, but this enzyme also experiences allosteric modulations (Harano et al., 1985; Murthy and Pande, 1987; Bezaire et al., 2004). CPT I is located on the inner side of the outer mitochondrial membrane and catalyses the conversion of acyl-CoA to fatty acylcarnitine (Kerner and Hoppel, 2000; Price et al., 2000). Quantitative RT-PCR is a reliable technique for measuring transcripts in small amounts of tissue (Spriewald et al., 2000). With this technique, multiple mRNAs can be assayed simultaneously in a relatively short period of time. Here we describe the standard procedure, optimized in our laboratory, to assess CPT I levels with β¬actin as an internal control in rainbow trout, and all the necessary controls to ensure a quantitative analysis. RNA Extraction and Reverse Transcription Total cellular RNA was isolated from liver of rainbow trout using RNX reagent (Cinnagen-Iran). To obtain cDNA, 1 µg of total RNA was subjected to reverse transcription polymerase chain reaction (RT-PCR) with MuLV reverse transcriptase using the RevertAidTM M¬MuLV Reverse Transcriptase Kit (Fermentase Life Science, Germany) and random hexamer primer. Reaction conditions in the reverse transcription step are mostly dependent on the enzyme and the primers of choice. Whereas other protocols to require the use of specific primers, we prefer to reverse transcribe the total RNA population with random hexamers so that different PCR analyses could be performed on the same cDNA sample

The interactive effect of water-borne cadmium and environmental hypoxia on common carp (Cyprinus carpio) metabolism

Regarding to the tight association between aquatic hypoxia and heavy metal contaminations in one hand and the role of both parameters on fish respiration, metabolism of carp could be assessed under single and mutual exposures to hypoxia and cadmium. Following measuring LC50-96h of cadmium (43.679 mg/l) for this species, 80 common carp were exposed to 10 different treatments, including control, acute (43.68 mg/l), sub-lethal (21.84 mg/l) and chronic (4.37 mg/l) cadmium as well as hypoxia for immediately (20% of saturation), 24h (40%) and 7 days (60%), and joint exposure of each similar treatment. By using of respirometer technique, we measured oxygen consumption rate in different time spans to calculate each individual standard metabolic rate (SMR), maximum metabolic rate (MMR), aerobic scope (AS), factorial aerobic scope (FAS) and critical oxygen tension (PCrit). Obtained data show that acute and sub-lethal cadmium treatments led to significant (P<0.05) increases in all metabolic indices in comparison with control group whilst the MMR and AS have been reduced (P<0.05) following hypoxia treatments. Combined treatments of hypoxia and cadmium led to reduce SMR and PCrit in all treatments and MMR, AS and FAS only in acute and sub-lethal treatments. In overall, hypoxia can act as a limiting stressor in carp while cadmium can account as a loading stressor

Determination of CPUA and distribution pattern of families Haemulidae, Nemipteridae and Ariidae in the Oman Sea

This trawl survey was carried out during 2013 for the stock assessment of families Haemulids, Nemipterids and Ariids in the Oman Sea. Sampling was carried out at five different stratum and depths. The highest value of CPUA of Haemulidae was estimated for Pomadasys stridens in “B” stratum (885.78 kg nm^-2), for Pomadasys kaakan at depths of 10-20 m (330.35 kg nm^-2), and for Nemipteridae it was estimated for Nemipterus japonicus in “D” stratum (1042.31 kg nm^-2) at 30-50 m depths (1734.97 kg nm^-2), and for Ariidae, it was estimated for Netuma thalassina in the stratum B (752.64 kg nm^-2) at 20-30 m depths (428.33 kg nm^-2). The highest biomass for Haemulidae was estimated in stratum B (320.53 ton) at 50-100 m depths (282.98 tons), and for Nemipteridae in “D” stratum (559.72 tons) and at depths of 30-50 m (604.04 tons), and for Ariidae it was estimated in “B” stratum (272.35 tons) and at 50-100 m depths (255.12 ton). Based on the results obtained, the highest species diversity for Haemulids was in “A” stratum at depths less than 50 m, while for Nemipterids it was similar in the total study area and different depth layers. Highest species diversity for Ariids were found in “A” and “D” strata at depth layers of 10-20 m and 30-50 m, respectively. In light of the fact that fishing efforts decreased during these years, our results illustrate that CPUA and biomass have ascending trends which indicate the relative stability of the stocks of these families

The sub-lethal effects of cadmium on some gill histopathology in the cultivated Sterlet (Acipenser ruthenus)

The sub-lethal effects of water-born Cadmium (Cd) on histopathological changes of gill tissues were investigated on Sterlet, Acipenser ruthenus. In this research, 60 fish (41.69 ± 0.95 g and 23.98 ± 0.14 cm) were randomly distributed in 4 experimental groups, each with 3 replicates. The fish were exposed to different concentrations of Cd for 21 days under semistatic experimental condition as: 0, 16, 32 and 64 µg/L Cd. At the end of the experiment, the gill tissues were fixed in Bouin's solution for histopathological studies. Qualitative and quantitative changes of the gill tissues were analyzed. Several qualitative changes including hyperplasia, hypertrophy of epithelial cells and epithelial edema as well as necrosis, rings and fusion of gill filaments and secondary lamellae were observed in all treated fish including control group. Quantitative analysis based on organ index (Iorg) showed significant increase in histopathological changes in the gill tissues coincided by elevation of Cd concentration in the water. However, there was no significant differences in the organ index between control and 16Cd group.The results of this study demonstrate severe histopathological changes in the gill tissues after exposing to sub-lethal concentration of water-born Cd under experimental condition. The results of this study might be used as a guildline for evaluation of histopahological changes being induced by heavy metals in natural or cultivated environment for sturgeon fish

Age, growth and reproduction of Chondrostoma regium (Heckel, 1843) from the Zayandeh Roud River, Iran

Age, growth and reproduction were investigated for Chondrostoma regium, collected monthly (November 2007- August 2008) from the Dimeh Spring region, Zayandeh Roud River, Iran. The maximum age of collected female and male fish were estimated based on scale increments as five and four years respectively. Von Bertalanffy growth equations were calculated as Lt=246[1-e^-0.206(t-0.034)], Wt=163.29[1-e^-0.206(t-0.034)]^2.77 for females and Lt=253.1[1-e^-0.206(t-0.175)], Wt=188.25[1-e^-0.206(t-0.175)]^2.99 for males. Size ranges of the male and female specimens were almost similar. The fork length range for females was 10.5-18.0 cm and 10.7-19.7 cm for males; and the weight range was 15.7-85.4 g in females, and 14.5-88.5 g in males. The female-male ratio was 1.8:1 (p<0.05), and for both sexes gonadal development and sexual maturity occurred in the second year of their life. Length-weight relationships revealed isometric growth pattern for males and a negative allometric growth for females. Monthly data of Gonadosomatic Index, and ovary condition suggested that spawning occurred during May and June. The mean absolute and relative fecundity was calculated as 2429±1512 (eggs/female) and 78.1±18.7 (egg/g), respectively, and fecundity was significantly correlated with both body weight (r^2=0.90) and fork length (r^2=0.84)

Effect of dietary wood betony, Stachys lavandulifolia extract on growth performance, haematological and biochemical parameters of common carp, Cyprinus carpio

A 6 week study was conducted to assess the effects of wood betony (WB), Stachys lavandulifolia extract on growth performance, hematological and biochemical parameters of common carp, Cyprinus carpio. Different levels of the WB extract (0, 2, 4 and 8 % weight per weight, W/W, 0WB, 2WB, 4WB and 8WB) in the diet were used. The results showed that final weight and weight gain were significantly improved by WB (p0.05). There were no significant differences in hemoglobin, hematocrit, mean erythrocytes of hemoglobin, mean erythrocyte volume, mean hemoglobin erythrocyte concentration and white blood cell (WBC) counts (p>0.05), while, red blood cells (RBC) counts showed significant declining trend by increasing the level of the plant extract from control to 8WB (p<0.05). Significant elevation in the levels of total protein, albumin and globulin and albumin/globulin ratio by increasing WB concentration in the diet were observed (p<0.05). Diet enriched by WB could decrease serum level of triglycerides and cholesterol in comparison with the control (p<0.05). Based on the results of this study, it could be concluded that feeding common carp with WB can improve growth and some immunity characteristics as well as lipid metabolism

Effect of dietary canola oil level on the growth performance and fatty acid composition of fingerlings of rainbow trout (Oncorhynchus mykiss)

This study evaluated the suitability of canola oil as a source of supplemental dietary lipid for rainbow trout. Triplicate groups of the 30 fingerlings held under identical culture conditions were fed twice daily by iso-nitrogenous, iso-calorific and iso-lipidic diets for eight weeks. Experimental diets consisted of 30.3% protein, 18.7 kJg^-1 energy and 16.7% lipid from fish oil (FO), canola oil (CO) and 1:1 blend of the two oils (FCO). Moisture, ash, protein, lipid, final body weight, condition factor, feed conversion ratio, survival and hepatosomatic indices were not affected by treatments. Specific growth rate and weight gain of fish reared on fish oil diet (FOD) and canola oil diet (COD) were significantly higher than those fed with the fish and canola oils diet (FCOD). Protein efficiency ratio was highest in fish fed with the COD. Whole body fatty acid compositions mirrored those of diet treatments. The highest amounts of HUFAs were detected in fish fed with FOD, which was significantly different from other treatments. In all treatments PUFAs/SFAs and n-6/n-3 ratios were higher than 0.45 and lower than 4, respectively. Our results indicate the fingerlings can be reared on diets in which FO has been replaced with CO, with no significant effects on fish growth performance

Epigenetic polypharmacology: from combination therapy to multitargeted drugs

The modern drug discovery process has largely focused its attention in the so-called magic bullets, single chemical entities that exhibit high selectivity and potency for a particular target. This approach was based on the assumption that the deregulation of a protein was causally linked to a disease state, and the pharmacological intervention through inhibition of the deregulated target was able to restore normal cell function. However, the use of cocktails or multicomponent drugs to address several targets simultaneously is also popular to treat multifactorial diseases such as cancer and neurological disorders. We review the state of the art with such combinations that have an epigenetic target as one of their mechanisms of action. Epigenetic drug discovery is a rapidly advancing field, and drugs targeting epigenetic enzymes are in the clinic for the treatment of hematological cancers. Approved and experimental epigenetic drugs are undergoing clinical trials in combination with other therapeutic agents via fused or linked pharmacophores in order to benefit from synergistic effects of polypharmacology. In addition, ligands are being discovered which, as single chemical entities, are able to modulate multiple epigenetic targets simultaneously (multitarget epigenetic drugs). These multiple ligands should in principle have a lower risk of drug-drug interactions and drug resistance compared to cocktails or multicomponent drugs. This new generation may rival the so-called magic bullets in the treatment of diseases that arise as a consequence of the deregulation of multiple signaling pathways provided the challenge of optimization of the activities shown by the pharmacophores with the different targets is addressed

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia.

ABSTARCT: Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. METHODS: Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. RESULTS: In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. CONCLUSIONS: This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis