Nicola King

The Effects of Natural Iron Fertilisation on Deep-Sea Ecology: The Crozet Plateau, Southern Indian Ocean

The addition of iron to high-nutrient low-chlorophyll (HNLC) oceanic waters stimulates phytoplankton, leading to greater primary production. Large-scale artificial ocean iron fertilization (OIF) has been proposed as a means of mitigating anthropogenic atmospheric CO2, but its impacts on ocean ecosystems below the photic zone are unknown. Natural OIF, through the addition of iron leached from volcanic islands, has been shown to enhance primary productivity and carbon export and so can be used to study the effects of OIF on life in the ocean. We compared two closely-located deep-sea sites (∼400 km apart and both at ∼4200 m water depth) to the East (naturally iron fertilized; +Fe) and South (HNLC) of the Crozet Islands in the southern Indian Ocean. Our results suggest that long-term geo-engineering of surface oceanic waters via artificial OIF would lead to significant changes in deep-sea ecosystems. We found that the +Fe area had greater supplies of organic matter inputs to the seafloor, including polyunsaturated fatty acid and carotenoid nutrients. The +Fe site also had greater densities and biomasses of large deep-sea animals with lower levels of evenness in community structuring. The species composition was also very different, with the +Fe site showing similarities to eutrophic sites in other ocean basins. Moreover, major differences occurred in the taxa at the +Fe and HNLC sites revealing the crucial role that surface oceanic conditions play in changing and structuring deep-sea benthic communities.

Mechanisms underlying the cardioprotective effect of L-cysteine

In many tissues the availability of l-cysteine is a rate-limiting factor in glutathione production, though this has yet to be fully tested in heart. This study aimed to test the hypothesis that supplying hearts with 0.5 mM l-cysteine would preserve glutathione levels leading to an increased resistance to ischaemia reperfusion.Left ventricular function was measured in isolated perfused rat hearts before, during and after exposure to 45 min global normothermic ischaemia. Control hearts received Krebs throughout, whilst in treated hearts 0.5 mM l-cysteine was added to the perfusate 10 min before ischaemia, and was then present throughout ischaemia and for the first 10 min of reperfusion. Reperfusion injury was assessed from the appearance of lactate dehydrogenase (LDH) in the effluent. In two separate groups of control and treated hearts, ATP and glutathione (GSH) contents were measured at the beginning and end of ischaemia.Hearts treated with 0.5 mM l-cysteine showed a significantly higher recovery of rate pressure product (16,256± 1288 mmHg bpm vs. 10,324± 2102 mmHg bpm, p < 0.05) and a significantly lower release of LDH (0.54± 0.16 IU/g wet weight vs. 1.44± 0.31 IU/g wet weight, p < 0.05) compared to controls. Also, the l-cysteine treated group showed significantly better preservation of ATP and GSH during ischaemia in comparison to control.These results suggest that the mechanisms underlying the cardioprotective effects of 0.5 mM l-cysteine may include: increased anaerobic energy production either directly or through reduced degradation of adenine nucleotides; direct scavenging of free radicals; and/or improved antioxidant capacity through glutathione preservation.

Aspartate transporter expression and activity in hypertrophic rat heart and ischaemia–reperfusion injury

This studys rationale was that the expression and activity of aspartate transporters in hypertrophied hearts might be different from normal hearts, which could affect the use of aspartate in myocardial protection of hypertrophied hearts. mRNA expression of system Xag− transporters in hearts from normal (Wistar Kyoto) and hypertrophied (spontaneously hypertensive rat) rats was investigated by RT‐PCR. EAAT3 protein expression in isolated cells and vesicles from normal and hypertrophied hearts was investigated by Western blotting. The same vesicles were also used to measure aspartate uptake. The effects of 0.5 mmol l−1 aspartate supplementation on cardiac performance during ischaemia–reperfusion were investigated in isolated and perfused hearts. Both normal and hypertrophied hearts expressed EAAT1 and EAAT3 mRNA. EAAT3 protein expression was significantly greater in cells and vesicles from hypertrophied hearts compared to normal hearts. The velocity (Vmax) of aspartate uptake was faster at 24.4 ± 2.2 pmol mg−1 s−1 in vesicles from hypertrophied hearts compared to 8.2 ± 0.8 pmol mg−1 s−1 (P < 0.001, t test, n= 6, means ±s.e.m.) in normal heart vesicles. The affinity (Km) was similar for both preparations. When recoveries were matched, 0.5 mmol l−1 aspartate addition reduced reperfusion injury and increased functional recovery of hypertrophied hearts but not normal hearts. This was associated with a greater preservation of ATP, glutamate and glutamine and less lactate production during ischaemia in aspartate‐treated hypertrophied hearts compared to all other experimental groups. These results suggest that increased aspartate transporter expression and activity in hypertrophy helps facilitate aspartate entry into hypertrophied cardiomyocytes, which in turn leads to improved myocardial protection.

Characteristics of l-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart

OBJECTIVE L-Aspartate is an important intermediary metabolite in the heart and has also been implicated in myocardial protection, but little is known about its transport across the cardiac sarcolemma. In this study we have tested the hypothesis that the high affinity sodium-dependent aspartate transporter, EAAC-1 is expressed in heart and have also characterised aspartate transport into the myocardium. METHODS Characteristics of L-[14C]aspartate uptake into rat heart were investigated using sarcolemmal vesicles and isolated myocytes. The expression of EAAC-1 in the two preparations was also investigated by western blotting. RESULTS The K(m) and V(max) of L-aspartate uptake was 9.78+/-0.7 microM and 1.17+/-0.27 pmol/mg/s in vesicles compared to 6.53+/-1.24 microM and 13.65+/-1.0 pmol/microl/s in cells. In vesicles, L-aspartate uptake was dependent on external sodium and internal potassium, and was rheogenic. In cells, L-aspartate uptake was also dependent on external sodium. Addition of unlabelled L- and D-aspartate and L-glutamate significantly inhibited L-[14C]aspartate uptake in both preparations but D-glutamate had no effect. An antibody to the aspartate transporter, EAAC-1 recognised a protein of appropriate size in both vesicles and cells. CONCLUSIONS L-aspartate uptake in heart is mediated by a high affinity sodium-dependent transporter. This is accompanied by the expression in heart of EAAC-1. The physiological significance of this transporter with respect to aspartate utilisation in the heart is discussed.

Effect of regular training on the myocardial and plasma concentrations of taurine and alpha-amino acids in thoroughbred horses.

SummaryExercise induces significant changes in the free intracellular amino acid pool in skeletal muscle but little is known of whether such changes also occur in cardiac muscle. In this study the effect of regular exercise on the size and the constituents of the free amino acid pool in the hearts and in the plasma of thoroughbred horses was investigated. The total free intracellular amino acid pool in the hearts of control horses was 30.9 ± 1.2μmol/g wet weight (n = 6). Glutamine but not taurine was present at the highest concentration (13.5 ± 0.9 and 7.7 ± 0.69μmol/g wet weight for glutamine and taurine respectively). As for the rest of the amino acids in the pool, only glutamate and alanine were present at levels greater than 1μmol/g wet weight (4.6 ± 0.25 and 1.7 ± 0.14 for glutamate and alanine respectively). The tissue to plasma ratio was highest for taurine at 155, followed by glutamate at 111, aspartate and glutamine at 37, alanine at 5.8 and ratios of less than 3 for the rest of the amino acids. The total free intracellular amino acid pool in the hearts of exercised horses was slightly but not significantly lower than control (28.1 ±1.1μmol/g wet weight, n = 6). Regular exercise increased the intracellular concentration of threonine, valine, isoleucine, leucine and phenylalanine but was only significant (p < 0.05) for threonine. This work has documented the profile of taurine and protein amino acids in the heart and in the plasma of thoroughbred horses and showed that in contrast to skeletal muscle, heart muscle does not show major changes in amino acids during regular exercise.

The intrinsic Cl–conductance of mouse kidney cortex brush-border membrane vesicles is not related to CFTR

Abstracts Brush-border membrane vesicles (BBMV) were prepared from whole Balb/c mice kidneys by a Mg2+ precipitation technique. The presence of an intrinsic Cl–conductance co-expressed with Na+/glucose cotransport was inferred by the anion dependence of [14C]glucose uptake and overshoot with inward Na+-anion gradients. In Na+-equilibrated conditions, an inside-negative membrane potential difference (p.d.) produced by an inward Cl–gradient alone was capable of driving intravesicular [14C]glucose accumulation. The apparent anion conductance had a selectivity of Br– = I– = Cl– >  F–>> gluconate, was inhibited by 0.5 mM 5-nitro-2- (3-phenylpropylamino)-benzoic acid (NPPB) but was unaffected by 0.5 mM 4,4′-diisothiocyanatostilbene 2,2′-disulphonate (DIDS). BBMV were isolated from mice in which the CFTR gene had been disrupted by a termination mutation (–/–) and compared with normal litter mates (+/+) and heterozygotes (–/+)[18]. [14C]Glucose uptake in NaCl media was significantly greater than glucose uptake in Na gluconate media for all three genotypes measured at 20 s: for homozygous –/– animals [14C]glucose uptake was increased by 2.80 ± 0.53 fold in Cl–media compared to gluconate media, n = 6; for wild-type +/+, by 2.16 ± 0.53 fold, n = 8; and for heterozygous +/– animals, by 2.17 ± 0.45 fold, n = 8. The observation of a Cl–-dependent component in BBMV isolated from homozygous –/– mutant animals shows that the chloride conductance in these vesicles cannot be due to cftr expression.

Effect of exercise training on liver function in adults who are overweight or exhibit fatty liver disease: a systematic review and meta-analysis

Objective Exercise training has been shown to have beneficial effects on liver function in adults overweight or with fatty liver disease. To establish which exercise programme characteristics were likely to elicit optimal improvements. Design Systematic review and meta-analysis of randomised, controlled trials. Data sources PubMed, CINAHL and Cochrane controlled trials registry searched (1966 to 2 October 2015). Eligibility criteria for selecting studies Exercise intervention, with or without dietary intervention, versus usual care in adults undertaking, exercise training, who were overweight, obese or exhibited fatty liver disease (non-alcoholic fatty liver disease or non-alcoholic steatohepatitis). Results We included 21 randomised controlled trials, totalling 1530 participants. Exercise intervention studies with total exercise programme workload >10 000 kcal produced significant improvements in intrahepatic fat, −3.46% (95% CI −5.20% to −1.73%), p<0.0001, I2=73%; effect size (standardised mean difference, SMD) −1.77 (−3.11 to −0.42), p=0.01, I2=77%. When data from only exercise studies were pooled, there was a reduction in fasting free fatty acids (FFAs) −74.15 µmol/L (95% CI −118.47 to −29.84), p=0.001, I2=67% with a large effect size (SMD) −0.94 (−1.36 to −0.52), p<0.0001, I2=0%. When data from only exercise studies were pooled, there was a significant reduction in insulin MD −1.88 UL (95% CI −3.43 to −0.34), p=0.02, I2=31%. The liver enzymes, alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transpeptidase, were not significantly altered with exercise. Conclusions Exercise training reduces intrahepatic fat and FFAs while increasing cardiorespiratory fitness. An aggregate exercise programme energy expenditure (>10 000 kcal) may be required to promote reductions in intrahepatic fat.

RT-PCR protocols

Part I: The RT-PCR Detective: Hunting Down the Best Method 1. Single Cell RT-PCR on Mouse Embryos: A General Approach for Developmental Biology Wolfgang Mann and Thomas Haaf 2. Poly(A) cDNA Real-Time PCR for Indicator Gene Measurement in Cancer Melissa Oliveira-Cunha, Ajith K. Siriwardena, and Richard J. Byers 3. Transcriptome Profiling of Host-Microbe Interactions by Differential Display RT-PCR W. F. Leong and Vincent T. K. Chow 4. Quantitative RT-PCR Methods for Mature microRNA Expression Analysis Stephanie D. Fiedler, Martha Z. Carletti, and Lane K. Christenson 5. Detection of Influenza A Virus Neuraminidase and PB2 Gene Segments by One Step Reverse Transcription Polymerase Chain Reaction Alejandra Castillo Alvarez, Victoria Boyd, Richard Lai, Sandy Pineda, Hans G. Heine, and Ross Barnard 6. Detection and Identification of CD46 Splicing Isoforms by Nested RT-PCR Anita Szalmas, Jozsef Konya, Istvan Sziklai, and Tamas Karosi 7. Simultaneous Detection of Bluetongue Virus RNA, Internal Control GAPDH mRNA, and External Control Synthetic RNA by Multiplex Real-Time PCR Frank Vandenbussche, Elise Vandemeulebroucke, and Kris De Clercq 8. Detection of West Nile Viral RNA from Field-Collected Mosquitoes in Tropical Regions by Conventional and Real-Time RT-PCR Ana Silvia Gonzalez-Reiche, Maria de Lourdes Monzon-Pineda, Barbara W. Johnson, and Maria Eugenia Morales-Betoulle 9. Detection of Antisense RNA Transcripts by Strand-Specific RT-PCR Eric C.H. Ho, Michael E. Donaldson, and Barry J. Saville 10. RT-PCR Amplification and Cloning of Large Viral Sequences Xiaofeng Fan and Adrian M. Di Bisceglie Part II: The RT-PCR Mathematician: Assessing Gene and RNA Expression 11. One-Step RT-LATE-PCR for mRNA and Viral RNADetection and Quantification Cristina Hartshorn and Lawrence J. Wangh 12. Changes in Gene Expression of Caveolin-1 after Inflammatory Pain Using Quantitative Real-Time PCR Fiza Rashid-Doubell 13. Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction Hongxin Fan and Ryan S. Robetorye 14. The Use of Comparative Quantitative RT-PCR to Investigate the Effect of Cysteine Incubation on GPx1 Expression in Freshly Isolated Cardiomyocytes Nicola King 15. The Renaissance of Competitive PCR as an Accurate Tool for Precise Nucleic Acid Quantification Lorena Zentilin and Mauro Giacca Part III: The RT-PCR Master Chef: Finding the Right Ingredients 16. Skeletal Muscle RNA Extraction in Preparation for RT-PCR Janelle P. Mollica 17. Reverse Transcription of the Ribonucleic Acid: A First Step in RT-PCR Assay Fadia Haddad and Kenneth M. Baldwin 18. Primer Design for RT-PCR Kelvin Li and Anushka Brownley 19. Hot Start PCR Natasha Paul, Jonathan Shum, and Tony Le 20. Real-time RT-PCR for Automated Detection of HIV-1 RNA During Blood Donor Screening Jens Muller

L-Leucine transport in rat heart under normal conditions and effects of a simulated hypoxia

Abstractl‐Leucine plays a central role in the regulation of protein metabolism in heart and has been implicated in myocardial protection, but little is known about the relationship between these phenomena and leucine transport across the cardiac sarcolemma. In this study we used sarcolemmal vesicles and ventricular myocytes isolated from rat heart to characterise l‐leucine transport under normal conditions and to investigate the effect of simulated hypoxia or inhibition of protein synthesis. The Km and Vmax of leucine uptake were 5.24 ± 0.65 mM and 1.43 ± 1.84 nmol min−1 mg−1 protein in vesicles compared to 2.17 ± 0.13 mM and 1.7 ± 0.76 nmol min−1 µl−1 intracellular space in cells. Transport was not dependent on Na+ or H+ gradients. In vesicles l‐leucine uptake was increased by trans‐stimulation, whilst inhibition was observed with classical system L substrates including 2‐aminobicyclo[2,2,1]‐heptane‐2-carboxylic acid (BCH) suggesting that this system mediated l‐leucine transport in heart.l‐Leucine uptake into isolated cardiac myocytes was inhibited after 20, 30 and 60 min of simulated hypoxia. This was not caused by reduced cell viability, although the cells underwent a rigor contracture. Inhibition of protein synthesis did not affect l‐leucine transport.

Effect of exercise therapy on established and emerging circulating biomarkers in patients with heart failure: a systematic review and meta-analysis

Background Biomarkers are important in the diagnosis, risk stratification and management of patients with heart failure (HF). The established biomarkers of myocardial stretch, brain natriuretic peptide (BNP) and amino (N) portion of BNP (NT-proBNP) have been extensively studied, and early analyses have demonstrated response to exercise training. Several other biomarkers have been identified over the last decade and may provide valuable and complementary information which may guide treatment strategies, including exercise therapy. Methods A systematic search of PubMed, EMBASE and Cochrane Trials Register to 31 October 2017 was conducted for exercise-based rehabilitation trials in HF. Randomised and controlled trials that reported biomarkers, BNP, NT-proBNP, soluble ST2, galectin-3, mid-regional atrial natriuretic peptide, mid-regional adrenomedullin and copeptin, were included. Results Forty-three studies were included in the systematic review, with 27 studies suitable for meta-analyses. Data pooling was only possible for NT-proBNP and BNP. Meta-analyses of conventional training studies demonstrated a statistically significant improvement in NT-proBNP (pmol/L); mean difference (MD) −32.80 (95% CI −56.19 to −9.42), p=0.006 and in BNP (pmol/L); MD −17.17 (95% CI −29.56 to −4.78), p=0.007. Pooled data of non-conventional training failed to demonstrate any statistically significant improvements. Conclusion Pooled data indicated a favourable effect of conventional exercise therapy on the established biomarkers, NT-proBNP and BNP; however, this was in contrast to a number of studies that could not be pooled. Limited evidence exists as to the effect of exercise training on emerging biomarkers.