Global transcriptional responses of the toxic cyanobacterium, Microcystis aeruginosa, to nitrogen stress, phosphorus stress, and growth on organic matter

Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller (5–22% of genes differentially expressed), transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.

苯丙氨酰tRNA合成酶的进化与结构域丢失

基因的复制、融合以及基因的水平转移是许多蛋白质包括氨酚t RNA合成酶(aminoacyl-tRNA synthetase , AARS)进化过程中的常见事件。然而作者研究的结果显示，苯丙氨酚 t RNA合成酶( phenylalanyl-tRNA synthetase , PheRS)的进化主要表现为 一些结构域的丢失;并且这种结构域的丢失不影响Phe RS的功能或活性。通常在生物从细菌到真核生物的进化过程中，其基因组的大小和基因的数目都有所增加，然而有趣的是，真核生物中Phe RS的结构域类型和数目都明显少于细菌的Phe RS oPhe RS通过结构域的丢失而进化的现象，似乎与某些AARS功能由多重专一性向单一专一性的演化有着“异曲同工”之妙。

不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响

干细胞冷冻保存是干细胞研究和临床应用中的必需技术.为提高兔胚胎干细胞在慢速冻存过程中的保存效果,比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol,EG)对兔胚胎干细胞冷冻保护效果.对冷冻复苏后的细胞进行台盼蓝染色,并研究其胚胎干细胞分子特性,结果表明DMSO比EG具有更好的冷冻保护效果.再在以10% DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine),细胞冷冻复苏后结果显示,谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用,使细胞存活率从71%提高到83.7%.当谷氨酰胺浓度为0、5、10、20、40 mmol/L分别加入防冻液中后,20 mmol/L的谷氨酰胺具有最佳的冷冻保护效果.以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为:在胚胎干细胞培养液中添加10% DMSO+20 mmol/L谷氨酰胺.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

腺苷蛋氨酸合成酶基因的克隆、改组与表达

S-Adenosyl-L-methionhie（SAM）是一种广泛存在于各种组织和细胞中的生物小分子，它在生物体内转甲基、转硫基和转氨丙基过程中起重要作用。SAM被广泛应用于治疗肝损伤、胆汁郁积和抑郁症等疾病。为改变当前酿酒酵母发酵胞内SAM积累量低的现状，本文以提高SAM胞内积累量为目的，综合应用现代基因工程技术，开展了SAM合成酶基因的克隆、改组与表达研究。在本试验中，我们通过PCR方法扩增得到三个SAM合成酶基因soml、samZ和metK，然后分别插入psE380载体并转化E．coliBL21。通过IPTG诱导可以获得各基因的蛋白质表达。三个目的基因被分别克隆进pYEsZ并转化酿酒酵母INVScl菌株，诱导表达后，工程菌株蛋白质表达量和胞内SAM积累量都有不同程度的提高。本论文建立了一种新的基因改组方法，可以将StEP和error-pronePCR有机的结合在一起，并成功的运用该方法对soml进行基因改组。经过一轮反应过程，蛋白质表达量和胞内SAM积累量均明显增加。对改组DNA序列分析表明，该基因具有8个点突变，导致4个氨基酸变异：I20L，G120S，I213L和A354S。为了进一步促进蛋白质表达，本文采用了一步长距离反向PCR方法，调整了起始密码子ATG和核糖体识别位点之间的间距，从而大幅度提高了蛋白质表达量和胞内SAM积累量。本论文建立了一种新的基因改组方法：分组一混合法，运用该方法可以将三种独立的基因改组过程整合进一个反应体系中。这一机制尤其适用于低同源性基因间的改组。采用本机制，仅需要一轮反应就实现了对samZ和metK的改组。通过对改组基因库的筛选，我们获得了一株SAM胞内积累量提高56.3％的酿酒酵母工程菌株。本论文建立起了一种改进的重叠PCR方法（MOE-PCR），该方法可以快速、高效的实现单个基因的多点定位突变。应用这一方法，仅需要一轮反应，就成功的实现了对soml基因8个稀有密码子的改造。

花椒主效化感物质分离鉴定及化感作用研究

近年来，随着对作物重茬障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。花椒（Zanthoxy piperitum.）为芸香科植物，是一种收益早、用途广、价值高的经济树种，是川西干旱河谷地区的重要经济作物，其连作障碍也倍受关注，系统研究花椒化感作用将有助于理解和最终解决花椒连作障碍问题。本文首先通过萃取、层析等方法分离花椒主效化感成分；通过外加不同浓度的花椒叶水浸液研究了对土壤氮素养分循环的影响；研究了花椒叶水浸液对苜蓿生理生化、光合作用、氮素养分吸收的影响，并对外施氮肥对这种化感影响的缓解作用做了研究；研究了花椒化感潜力对全球变化——UV-B增强辐射的响应。主要研究结果如下： 1．用不同极性的有机溶剂对花椒叶水浸液浓缩浸膏萃取、柱层析，结合生物活性检测，分离得到主效化感作用组分的一种化感物质——对甲氧基苯酚。采用该物质纯品进行生物活性检测，证明其具有化感作用。 2．花椒叶水浸液处理土壤30天后，土壤硝态氮、铵态氮、无机氮（硝态氮＋铵态氮）与对照相比，随着花椒叶水浸液浓度的增加呈现降低的趋势，其中土壤铵态氮含量显著降低，而硝态氮含量的变化则不显著，无机氮含量也显著降低。土壤脲酶和蛋白酶的活性与无机氮含量的变化趋势相同。随着花椒叶水浸液浓度的增加，氨化细菌数量显著降低，固氮菌的数量变化不显著，硝化细菌和反硝化细菌数量有减少的趋势。60天后，硝态氮含量、铵态氮含量、无机氮随水浸液浓度增加的变化趋势与30天时相似；随着花椒叶水浸液浓度的增加，氨化细菌、固氮菌的数量显著减少，硝化细菌数量、反硝化细菌数量仍呈减少趋势；土壤脲酶、蛋白酶活性与第30天的变化趋势相同。第60天与第30天的结果相比，相同水浸液浓度处理的硝态氮、铵态氮、无机氮均有下降的趋势，但除了25g.L－1水浸液处理的外，其它相同浓度的处理间差异均不显著；除了12.5 g.L的处理外土壤脲酶活性均呈增强的趋势；蛋白酶活性都有不同程度的增加；花椒叶水浸液处理的土壤硝化细菌和反硝化细菌数量呈增加趋势。 3．随着花椒叶水浸液浓度的增加，显著抑制了苜蓿根长、地上地下生物量、叶绿素含量、叶片中可溶性蛋白的含量，净光合速率。苜蓿体内四种抗氧化酶（POD、SOD、CAT、APX） 活性随着水浸液浓度的增加而降低，而丙二醛含量则增加。苜蓿氮初级同化相关酶硝酸还原酶（NR）、谷氨酰合成酶(GS)、谷氨酸脱氢酶（GDH）的活性随着水浸液浓度的增加受到不同程度的影响。总的来说，苜蓿硝酸还原酶、谷氨酰合成酶的活性受到抑制，而谷氨酸脱氢酶活性的变化则比较复杂，根呈先降低后增加的趋势，叶片则无显著变化。外施两种不同浓度的硝酸铵氮肥后，对12.5、25 g.L－1花椒叶水浸液处理的苜蓿化感作用有显著的缓解作用，表现在株高、生物量、光合作用等方面，大多达到与对照（0 g.L－1）未施氮肥无显著差异的水平，而对50 g.L－1水浸液处理的苜蓿幼苗，虽有一定的缓解作用，但这种作用均未达到与对照（0 g.L－1）未施氮肥时无显著差异的水平。 4． UV-B增强辐射处理花椒后，花椒的化感潜力显著增强。花椒叶片内UV-B吸收物质的含量和总酚含量均显著增加。 In recent years, with profound research on the reasons of continuous cropping obstacles, allelopathy received increasing attention to many scholars at home and abroad. Zanthoxy bungeanum as a Rutaceae plant is a high economic value species which gains early and uses widely. Zanthoxylum is an important economic crop in the arid valley of western Sichuan region, and its not even has received much concern for the continuous cropping obstacles. The systematic study of allelopathy of Zanthoxylum will contribute to the understanding and final settlement of this issue. The major allelopathic composition was separated through the extraction, chromatography combined with other methods. The impact on soil nutrient cycling was also studied through the addition of different concentrations of water extracts of Zanthoxylum. Furthermore, the effects of water extracts of Zanthoxylum leaves on alfalfa leaf physiological and biochemical indexes, photosynthesis, soil enzymes and nutrient uptake of nitrogen and the mitigation of allelopathy through using external fertilizer were studied to put forward scientific resolvent for Zanthoxylum continuous cropping obstacles .The response of allelopathic potential of Zanthoxylum to global change - UV-B enhanced radiation was studied . The main findings are as follows: 1. Through extraction with different polar organic solvents on concentrated water extract of Zanthoxylum leaf and then using column chromatography combined with detection of biological activity, one of the main allelopathic components- methoxy-phenol was isolated. The biological activity testing of the pure material of methoxy-phenol proved that it does have allelopathic potential. 2. Thirty days after treating soil with water extract of Zanthoxylum leaf, as compared with the control, the contents of soil nitrate, ammonium, nitrate plus ammonium nitrogen showed a trend of decrease with the increase of the concentration of water extract whereas the content of ammonium nitrogen showed a significant reduction, and the content of nitrate did not change significantly, the content of nitrate plus ammonium nitrogen also showed a significant (P <0.05) redction. The activity of soil urease and protease showed the same trend as the content of nitrate nitrogen plus ammonium nitrogen. With the increase in the concentration of water extract, the number of ammonification bacteria significantly reduced but nitrogen-fixing bacteria did not change significantly and there was a decreasing trend in the number of nitrifying bacteria and denitrifying bacteria. Sixty days after the treatment, with the increase in solution concentration of water extract of Zanthoxylum leaf, the content of nitrate、 ammonium nitrogen, nitrate plus ammonium nitrogen showed a similar change trend to 30 days’; the number of ammonification bacteria, nitrogen-fixing bacteria significantly reduced ; the number of nitrifying bacteria, denitrifying bacteria was still an downward trend; the activity of soil urease and protease showed the same trend as the 30th days’. Compared to the results of the 30th days’, the content of nitrate, ammonium, nitrate plus ammonium nitrogen showed a decrease trend between the treatment of same concentration, but there was no significant difference except the treatment of 25g.L-1 between the same concentration; the activity of soil urease showed enhanced trend except the treatment of 12.5 g.L-1; the activity of protease increased to varying degrees; the number of ammonification bacteria、 nitrifying bacteria and denitrifying bacteria were growing while nitrogen-fixing bacteria reduced.. 3. With the increase of the concentration of water extract of Zanthoxylum leaf, the water extract significantly inhibited the root length, aboveground biomass, content of chlorophyll and soluble protein in leaf and net photosynthetic rate. The activity of four antioxidant enzymes (POD, SOD, CAT, APX) reduced with the increase in concentration of the water extract but the content of MDA increased. The activity of enzymes related to primary nitrogen assimilation such nitrate reductase (NR), glutamyl synthetase (GS), glutamate dehydrogenase (GDH) were subject to different degrees with an increase in the concentration of water extracts. In general, the activity of nitrate reductase, glutamyl synthetase were inhibited, while change in the activity of glutamate dehydrogenase was more complex. The activity of glutamate dehydrogenase in leaf was first reduced and then increase,but did not change significantly in root. After using two external different concentrations of nitrogen fertilizer, there was a significant mitigation in inhibiton in plant height, biomass, photosynthesis, etc. in the treatment of 12.5,25 gL-1 of water extract of Zanthoxylum leaf, and most of these indexes showed no significant difference with the control (0 g.L-1, no external fertilizer was added) .Although there showed a certain degree of ease in the treatment of 50 g.L-1 , there was still a significant difference compared with the control (0 gL-1) in which no external fertilizer was used. 4.The allelopathic potential of Zanthoxylum positively responded to enhanced UV-B significantly. The content of UV-B absorbing compounds and the total phenol also significant increased.

A quick isolation method for mutants with high lipid yield in oleaginous yeast

A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 mu mol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol-chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism.

氨酰-tRNA合成酶结构域的进化研究

氨酰-tRNA合成酶(Aminoacyl-tRNA synthetases, aaRS)是一类在蛋白质生物合成中具有重要作用的酶，它可以活化氨基酸，并与相应的tRNA相识别，使得基因序列能够被精确的翻译成蛋白质序列，保证了生命体的严谨性和多样性。通常，每一类aaRS都包含有一个催化核心结构域(Catalytic central domain, CCD)和一个结合反密码子的结构域(Anticodon-binding domain, ABD)。大量研究显示，细菌与真核生物中的许多aaRS在一些细菌与真核生物中的基因进化机制与模式、氨酰化途径、结构与功能的进化模式等方面往往有着明显的差异。通过对这些差异的深入研究，对于理解蛋白质的结构、功能的进化将是非常有帮助的。虽然，造成这些差异的本质，目前仍不清楚，但是，所有的这些差异似乎提示，在细菌与真核生物的一些基本生命活动过程中的某些方面，可能还存在着目前尚未被人们所认识到的较大差异。 甘氨酰-tRNA合成酶(Glycyl-tRNA synthetase，GlyRS)在基因组中存在着两种寡聚体形式，即α2β2四聚体和α2二聚体。本研究的结果显示，四聚体和二聚体GlyRS的ABD并不同源，而它们的CCD却具有共同的起源。在进化过程中，由于基因的融合，二聚体GlyRS的ABD融合到α亚基上CCD后的C-末端，而四聚体GlyRS的ABD则加在了β亚基的C-末端。通常，同一物种中只存在一种寡聚体形式的GlyRS，但是在Magnetospirillum magnetotacticum基因组中同时存在GlyRS的两种寡聚体形式，并有多个同源的结构域，而这些同源的结构域很可能来源于不同的基因组。二聚体GlyRS存在于细菌、古细菌和真核生物中，而四聚体GlyRS仅在大多数细菌中发现。在从细菌到真核生物的进化过程中，GlyRS可能经历了一个复杂的进化历程。频繁的基因丢失和获得事件导致了GlyRS分布的差异。水平基因转移是四聚体GlyRS进化的一个主要因素。大量的细菌基因水平转移导致四聚体GlyRS基因可在植物中表达，而在动物中形成假基因。 通常，由于aaRS-I和aaRS-II具有不同的结构和催化机制，它们被认为在进化上没有联系。虽然，苯丙氨酰-tRNA合成酶(phenylalanyl-tRNA synthetase, PheRS)属于aaRS-II，但它的催化机制却类似于aaRS-I。结构域的进化分析表明，细菌、古细菌和真核生物的PheRS具有明显不同的结构，因而导致从细菌到真核生物的进化过程中，PheRS和 tRNAPhe间的识别机制发生了变化。序列分析表明，PheRS的结构域(包括CCD、ABD及其它结构域)与aaRS-I的某些结构域同源，因此，在进化上，PheRS是aaRS-II与aaRS-I之间联系的纽带。这些结果表明，在进化的过程中，aaRS-I和aaRS-II可能是由同一个共同的祖先CCD经过可变剪接和插入演化而来的，结构域间的不同组合导致aaRS-I和aaRS-II在结构和催化机制上的显著差异。

A chemiluminescence optical fiber glucose biosensor based on co-immobilizing glucose oxidase and horseradish peroxidase in a sol-gel film

An optical fiber bienzyme sensor based on the luminol chemiluminescent reaction was developed and demonstrated to be sensitive to glucose. Glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized by microencapsulation in a sol-gel film derived from tetraethyl orthosilicate(TEOS). The calibration plots for glucose were established by the optical fiber glucose sensor fabricated by attaching the bienzyme silica gel onto the glass window of the fiber bundle. The linear range was 0.2-2 mmol/L and the detection limit was approximately 0.12 mmol/L. The relative standard deviation was 5.3% (n = 6). The proposed biosensor was applied to glucose assay in ofloxacin injection successfully.

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells. (C) 2003 Elsevier Science B.V. All rights reserved.

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve

Marine sponge cell culture is a potential route for the sustainable production of sponge-derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96-well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett-Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.

Diurnal variation in uptake and xylem contents of inorganic and assimilated N under continuous and interrupted N supply to Phleum pratense and Festuca pratensis

J. H. Macduff and A. K. Bakken. (2003). Diurnal variation in uptake and xylem contents of inorganic and assimilated N under continuous and interrupted N supply to Phleum pratense and Festuca pratensis. Journal of Experimental Botany, 54 (381) pp.431-444 Sponsorship: BBSRC / Norwegian Crop Research Institute RAE2008

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.